Mesenchymal Stem Cell-Derived Extracellular Vesicles to the Rescue of Renal Injury

Acute kidney injury (AKI) and chronic kidney disease (CKD) are rising in global prevalence and cause significant morbidity for patients. Current treatments are limited to slowing instead of stabilising or reversing disease progression. In this review, we describe mesenchymal stem cells (MSCs) and their constituents, extracellular vesicles (EVs) as being a novel therapeutic for CKD. MSC-derived EVs (MSC-EVs) are membrane-enclosed particles, including exosomes, which carry genetic information that mimics the phenotype of their cell of origin. MSC-EVs deliver their cargo of mRNA, miRNA, cytokines, and growth factors to target cells as a form of paracrine communication. This genetically reprograms pathophysiological pathways, which are upregulated in renal failure. Since the method of exosome preparation significantly affects the quality and function of MSC-exosomes, this review compares the methodologies for isolating exosomes from MSCs and their role in tissue regeneration. More specifically, it summarises the therapeutic efficacy of MSC-EVs in 60 preclinical animal models of AKI and CKD and the cargo of biomolecules they deliver. MSC-EVs promote tubular proliferation and angiogenesis, and inhibit apoptosis, oxidative stress, inflammation, the epithelial-to-mesenchymal transition, and fibrosis, to alleviate AKI and CKD. By reprogramming these pathophysiological pathways, MSC-EVs can slow or even reverse the progression of AKI to CKD, and therefore offer potential to transform clinical practice.


Pathophysiology of AKI and CKD
Acute kidney injury (AKI) and chronic kidney disease (CKD) are established as global health burdens, with a prevalence of one in ten adults for CKD [1]. AKI refers to a sudden deterioration in renal function, resulting in increased plasma creatinine and blood urea nitrogen (BUN) and/or declining urine output within hours to days [2][3][4]. The most common causes are pre-renal with hypovolaemia, ischaemia, toxic injury, and sepsis, leading to oxidative stress, inflammation, apoptosis, and necrosis of tubular epithelial cells (TECs) [5]. In response, the remaining TECs proliferate and differentiate so recovery occurs over a few weeks [5][6][7].
However, these nephroprotective mechanisms can be overwhelmed and there is a 25% risk of progression to CKD [8], and a 50% increase in 10-year mortality, particularly from coronary events [9,10]. The most common cause of CKD is diabetes, followed by glomerulonephritis, hypertension, and polycystic kidney disease, with smoking, a family history of kidney failure, obesity, ≥60 years old, and being of Aboriginal or Torres Strait Islander origin contributing to the overall risk [1,11]. In Type 2 diabetes mellitus (T2DM), hyperglycaemia induces oxidative stress and inflammation, which leads to maladaptive repair by fibroblasts and podocytes, and reduction of peritubular endothelial capillary (PTC) networks [5,12,13]. A notable mechanism is the epithelial-to-mesenchymal transition Exosomes are derivatives of the endosomal compartment, secreted into the environment when multivesicular bodies (MVB) fuse with the plasma membrane [44,47,48]. Given the difficulties in differentiating the subtypes of EVs by their size, the International Society for Extracellular Vesicles (ISEV) prefers the collective term of "EVs" to define particles released from cells that are bounded by a lipid bilayer and cannot replicate [49]. The mechanism by which EVs are secreted from MSCs and mediate paracrine communication. MSCs can be harvested from multiple tissue sources (bone marrow, liver, kidney, adipose tissue, urine, umbilical cord blood, Wharton's jelly, placenta). EVs are membrane-enclosed particles classified into three categories: apoptotic bodies, microvesicles, and exosomes. For example, exosomes are secreted when MVB fuse with the plasma membrane and are characterised by surface expression of CD9, CD63, and CD81. They deliver a cargo of mRNA, miRNA, ncRNA, cytokines, chemokines, and growth factors to nearby injured cells. EVs utilise specific receptors or membrane fusion to enter target cells and the delivered material alters gene expression and cellular fate. This reprograms pathophysiological pathways, such as proliferation, apoptosis, angiogenesis, oxidative stress, and immunomodulation. MSC: mesenchymal stem cell; MVB: multivesicular bodies; EVs: extracellular vesicles; CD: cluster of differentiation; ncRNA: non-coding RNA.
EVs carry a cargo reflecting the phenotype of their cells of origin [50], which includes cytokines, chemokines, growth factors, mRNA, miRNA, and other non-coding RNA [28,51,52]. The collection of surface proteins, originating from the endosomal pathway, distinguishes exosomes from MVs and includes tetraspanins (CD9, CD63, CD81), heat shock protein (HSP) 70, ALIX, and tumour suppressor gene 101 [27,40,49]. EVs use specific receptors or membrane fusion to enter target cells and deliver their contents as a form of paracrine communication [53]. Consequently, the delivered material modifies the phenotype of recipient cells by altering gene expression, stimulating transcription, inducing phenotypic switches, or determining cellular fate, self-renewal, and differentiation [29,52]. At the cellular level, this reprograms pathophysiological pathways, such as proliferation, apoptosis, oxidative stress, angiogenesis, and immunomodulation to promote tissue regeneration [4,45,50,54]. Therefore, this review will summarise the methodologies for isolating exosomes, the different clinical applications of MSC-EVs and, more specifically, their regenerative capacity in treating animal models of AKI and CKD.

Methodologies of Exosome Isolation
The therapeutic use of exosomes requires the development of methodologies that isolate exosomes of suitable quantity and quality from MSCs [43]. Exosomes can be collected from various biofluids, including plasma, serum, urine, and saliva [55] with conditioned cell culture media being the most widely used material [56]. Current isolation protocols are based on the physical properties of density and size, or chemically through the surface interactions with proteins [57] (Table 1). However, the methods are tedious and non-specific and there is no consensus on a gold standard of isolation [58], with differential ultracentrifugation chosen by 80% of researchers [56]. There is no set characterisation of the three populations of EVs by size, so most methods isolate a heterogeneous population [40,45,59,60]. Further standardisation is required.

Invitrogen Precipitation
Affinity-Based

Principle
Based on size and sedimentation rate by successive centrifugation at increasing speed and duration [61,62] Based on density upon flotation or pelleting [45,63] Based on separating sample molecules relative to pore size of chromatography gel column [57,64] Compound polymer-based precipitation [63] Affinity interaction between surface protein, sugar, or lipids, with antibodies coated on magnetic beads [48,57,63,65]

Macular Degeneration
An ongoing Phase I trial (NCT03437759) is investigating the healing capacity of umbilical-MSC-exosomes to repair areas of large and refractory macular injury [77]. A single dose of 20 µg or 50 µg exosomes will be applied around the injured macular area in 44 patients and the visual outcome will be followed up six months later. Pre-clinical studies propose that MSC-exosomes suppress inflammation and inhibit apoptosis by downregulating MCP-1, a key chemoattractant for monocytes [76].

Cancer
Tumour cells are known to secrete pathogenic exosomes to facilitate paracrine communication in the tumour microenvironment and promote tumour growth, invasion, metastasis, and drug resistance [90]. Many clinical trials have focused on the role of exosomes as diagnostic (NCT04394572) [91] or prognostic (NCT04288141) [92] indicators in cancer. However, MSC-exosomes may also be therapeutic in targeting cancers with known driver mutations. For example, MSC-exosomes engineered to carry siRNA specific to the oncogenic KrasG12D mutation (iExosomes) have successfully supressed pancreatic ductal adenocarcinoma in mice [93]. In a current Phase I trial (NCT03608631), these iExosomes will be delivered intravenously on days 1, 4, and 10 and repeated every 14 days for three courses of treatment for patients with Stage IV pancreatic ductal adenocarcinoma, harbouring the KrasG12D mutation [94].

Alzheimer's Disease
The ability of MSC-EVs to cross the blood brain barrier means they could treat neurodegenerative diseases, such as AD [42]. EVs can directly internalise β-amyloid for lysosomal clearance [81], or transfer an insulin-degrading enzyme [95] or small interfering RNA [96] to reduce β-amyloid production, which is considered pathogenic in AD. A phase I/II trial (NCT04388982) is currently investigating the safety and efficacy of 5 µg, 10 µg, or 20 µg of allogenic adipose-MSC-exosomes administered to patients with AD twice a week for three months [82].

Ischaemic Stroke
As a result of shared risk factors, patients with Stage 4 CKD are twenty times more likely to die prematurely from cardiovascular events than progressing to end-stage kidney failure [9,20]. Preclinical studies in mice showed that miR-124-enriched exosomes induced neurogenesis, protected against ischaemic injury, and prevented post-ischaemic immunosuppression [84]. In an ongoing Phase II/III trial (NCT03384433), BM-MSC-exosomes, loaded with miR-124, will be administered to the ischaemic area one month following the stroke and the patient's neurological outcome will be assessed one year later [83].

ARDS and COVID-19
The anti-inflammatory effect of MSC-exosomes is being investigated in coronavirus (SARS-CoV-2) pneumonia and acute respiratory distress syndrome (ARDS), where only supportive care exists [87]. A few clinical trials are currently investigating the efficacy of aerosol inhalation of allogenic adipose-MSC-exosomes in the treatment of ARDS over seven days (NCT04602104) [88] and intravenous delivery of an escalating dose of MSC-exosomes over five days in COVID-19 pneumonia (NCT04798716) [89]. The mechanism by which EVs elicit their immunomodulatory effects and whether they would be suitable therapeutic candidates is yet to be determined [97].

Nephroprotective Role of MSC-EVs in AKI
Multiple animal models have demonstrated that MSC-EVs can ameliorate AKI induced by cisplatin, glycerol, gentamicin, or ischaemic-reperfusion injury (IRI) [43,98,99]. Given the heterogeneous nature of MSC-EVs, it currently remains unknown which sub-types provide renoprotection, so the efficacy of MVs, MPs, and exosomes in rodent AKI will all be discussed (Table 2).

Tubular Proliferation and Dedifferentiation
Biodistribution analyses illustrate that MSC-EVs specifically accumulate at the site of injury and their cargo of growth factors determines regenerative capacity [100]. In glycerol- [101] or cisplatin-triggered [102] AKI and IRI [53,103,104], MSC-MVs delivered mRNA of the mesenchymal phenotype or IGF-1 receptor [102] to proximal TECs and this induced expression of hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP). This promoted proliferation and dedifferentiation of proximal TECs. Similarly, BM-MSC-EVs, enriched with pro-regenerative miRNA (miR-10a, miR-486), induced TEC proliferation, reduced BUN, creatinine, and proteinuria, and improved renal function following glycerol-triggered AKI [105]. Additionally, activation of the ERK1/2 pathway and downregulation of p38 MAPK signalling was attributed to increased cell proliferation and the reversal of cisplatin-mediated damage in kidneys treated with umbilical-MSCexosomes [106] and BM-exosomes [107,108].

Angiogenesis
The horizontal transfer of proangiogenic factors, such as vasculogenic growth factor (VEGF-A), IGF-1, and basic fibroblast growth factor (bFGF) from MSC-EVs to resident cells mediates nephroprotection by EVs [108,111,113,124]. The downregulation of HIF-1α led to increased density and perfusion of renal capillaries, thereby reducing hypoxia [111].

Anti-Oxidation
During ischaemia, ATP levels rapidly fall, whilst intracellular calcium, protons, and reactive oxygen species (ROS) levels and lactic acid rise [125,126]. Increased mitochondrial membrane permeability and release of lysosomal enzymes cause breakdown of TECs. BM-MSC-EVs reduced ischaemic damage in isolated rat kidneys by upregulating enzymes involved in cellular metabolism (Calbindin1) and ion membrane transport (Slc16a1, vacuolar H + -ATPase d2 subunit) [74]. Calbindin1 sequesters excess calcium and reduces ROS and apoptosis [127]. Slc16a1 encodes for monocarboxylate transporter 1 and exports accumulated lactic acid [128]. Additionally, H + -ATPase pumps protons across the cell membrane and this reduces intracellular acidosis [129]. Therefore, EV-treated kidneys had lower glucose but higher pyruvate levels compared to ischaemic kidneys [74], indicating the important anti-oxidant activity of EVs [126].
The anti-oxidant activity of EVs may involve upregulation of nuclear factor E2-related factor (Nrf2) [130], which is a transcription factor binding anti-oxidant response elements and improves the expression of ROS scavenging enzymes, such as superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) [131]. Wharton's jelly MSC-EVs [130] and -MVs [119] alleviated oxidative stress in rats with unilateral kidney ischaemia and IRI, respectively, by downregulating NOX2 expression, which is a NADPH oxidase generating ROS. The authors hypothesised that miRNA delivered by EVs activated Nrf2 [130] or suppressed NOX2 expression [119]. Another IRI study supports this mechanism where human placenta-MSC-EVs delivered a cargo of miR-200a-3p to TECs [132]. The miRNA downregulated Keap1, freeing Nrf2 for nuclear translocation and promoted SOD2 expression. This reinforced antioxidant defence, increased ATP production, and protected TECs from mitochondrial fragmentation.
Melatonin is a strong scavenger of ROS and exosomes derived from MSCs conditioned with melatonin reduced oxidative stress within three days of IRI [133]. Exosomes carried RNA that downregulated expression of ROS such as malondialdehyde, HIF-1α, and NOX2, and upregulated anti-oxidant molecules (HO-1, SOD, catalase, glutathione peroxidase) [113].
As mentioned earlier, Wharton's jelly MSCs exert greater immunomodulatory activity than other sources of MSCs [35,36]. A single injection of Wharton's jelly MSC-MVs delivered miR-15a/-15b/-16 to rats with IRI and this suppressed CX3CL1 expression and CD68+ macrophage infiltration [110,118]. Furthermore, surface expression of CCR2 by exosomes could sequester its extracellular ligand, CCL2, and interfere with macrophage recruitment in IRI [134]. Additional studies demonstrated Wharton's jelly MSC-EVs decrease NK cell infiltration in ischaemic kidneys through downregulation of CX3CL1 and TLR2 expression [138]. This immunomodulation was preserved in rats with a splenectomy, indicating the spleen was not necessary for EVs to mediate renoprotection, unlike MSCs.

Anti-Fibrotic Effect of MSC-EVs in CKD
Renal glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of diabetic nephropathy and indeed all types of CKD [4]. MSC-EVs promote tissue regeneration by targeting kidney fibrosis, reducing tubular atrophy and inflammation, and facilitating angiogenesis to abrogate pathogenic insults in CKD (Table 3).

Reduce Tubular Atrophy
EVs exhibit anti-apoptotic activity to prevent the transition of AKI into CKD [109,145,153]. Six months following treatment with umbilical-MSC-MVs, rats with IRI showed dwindling tubular atrophy, improved functioning, and decreased glomerular ECM accumulation and fibrosis [104]. Reduced-to-absent tubular atrophy and repaired renal morphology were also observed in mice with 5/6 subtotal nephrectomy treated with MSC-MVs [153]. Furthermore, BM-MSC-EVs reduced degeneration, vacuolisation, tubular cyst formation, and atrophic changes of proximal TECs in mice with T1DM, T2DM [16], or cyclosporin nephrotoxicity [154].
A high cholesterol/fructose diet induces Metabolic Syndrome that not only increases the risk of CKD progressing to end-stage kidney failure [156], but also hinders the proliferative and differentiation potential of MSCs [157]. In a swine model of unilateral renovascular disease and Metabolic Syndrome, autologous adipose-MSC-EVs, enriched with pro-angiogenic factors (VEGF-A,C, VEGF receptor, angiopoietin-like 4, HGF), were internalised by tubular and endothelial cells within four weeks and improved cortical microvascular density, renal blood flow, and GFR [158].

Anti-Inflammatory
Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed by TECs and PTCs to support the recruitment of inflammatory cells into injured kidneys [159]. In T1DM and T2DM mice, BM-MSC-exosomes reduced expression of ICAM-1 in PTCs and reversed infiltration of dendritic cells, thereby preventing the development of diabetic nephropathy [16]. Additionally, BM-MSC-EVs downregulated CCL3 and hindered recruitment of macrophages and T cells [54,160]. Another study demonstrated that adipose-MSC-EVs delivered miR-26a-5p to inhibit TLR4 and the NF-κB/VEGFA inflammatory pathway, thereby alleviating diabetic nephropathy [161]. BM-MSC-EVs reduced TNF-α expression and inflammation, leading to an improvement in CKD outcomes [16].
TGF-β induces gene expression of forkhead box-P3 (FoxP3) to create a population of regulatory CD4+ T cells (Tregs) that police excessive inflammation and this can be utilised to ameliorate CKD [162]. For example, MSC-EVs, harvested from lean pigs, upregulated TGF-β expression and induced Treg differentiation in pigs with Metabolic Syndrome and unilateral renal artery stenosis, thereby decreasing inflammation and tubulointerstitial fibrosis [163]. Lean-EVs shifted the balance of macrophages from a pro-inflammatory M1 to anti-inflammatory M2 phenotype, and reduced the numbers of cytotoxic CD8+ T cells and IL-1β expression. By contrast, MSC-EVs, derived from pigs with Metabolic Syndrome, failed to alleviate CKD. This indicates the importance of the source and phenotype of MSC-EVs, where Metabolic Syndrome altered the cargo of 19 mitochondria-related miRNAs and therefore impaired the therapeutic efficacy of EVs [164].

Biological Cargo Carried by MSC-EVs to Alleviate AKI and CKD
MSC-EVs can be engineered to carry different biomolecules and target the interplay of signalling pathways responsible for AKI and CKD ( Figure 2).

YAP
YAP is a transcription factor in the Hippo signalling pathway and co-localises with α-SMA in the nucleus of TECs to promote fibrosis through an unclear mechanism [176]. Umbilical-MSC-exosomes delivered casein kinase 1δ and E3 ubiquitin ligase β-transducin repeats-containing protein to trigger ubiquitination and degradation of YAP in TECs [166]. This reduced collagen and ECM deposition and attenuated fibrosis associated with UUO.

Oct-4
Oct-4 is known as one of the four transcription factors capable of reprogramming fibroblasts into induced pluripotent stem cells (iPSCs) [177] and it can downregulate Snail and the EMT [178]. Umbilical-MSC-EVs overexpressing Oct4 reduced apoptosis, promoted TEC proliferation, and rescued mice with IRI from fibrosis within two weeks [117].

SP1
MSC-EVs from human iPSCs can deliver sphinganine-1-phosphate 1 (SP1) to PTCs to directly bind the promoter region of sphingosine kinase 1 [122]. This increased SP1 expression and inhibited necroptosis in rats with IRI, elucidating a novel mechanism of EVs in nephroprotection.

Sox-9
Sox-9 is a transcription factor of the sex-determining region Y box family and may repair injured TECs [179]. Adipose-MSCs-exosomes upregulated Sox9 and prevented TGF-β1-induced transformation of TECs into a pro-fibrotic phenotype in mice with IRI [168]. Increased Sox9 stimulated TEC proliferation, attenuated AKI, and protected the development of tubulointerstitial fibrosis. Another study used two-photon microscopy to track human placenta-MSC-EVs migrating to kidneys injured by IRI. MSC-EVs promoted Sox9 activation in TECs to stimulate regeneration and reduce fibrosis within four weeks [142].

SIRT1
Sirtuin 1 (SIRT1) is an NAD + -dependent deacetylase of the sirtuin family that is expressed by various kidney cells during stress and inhibits inflammation, apoptosis, and fibrosis [180]. In sepsis-induced AKI, adipose-MSC-exosomes inhibited NF-κB-mediated transcription of pro-inflammatory cytokines in the SIRT1 pathway and reduced immune cell infiltration and apoptosis [141]. Furthermore, glial cell line-derived neurotrophic factor (GDNF) was transfected into adipose-MSCs, and their exosomes ameliorated fibrosis in mice with UUO [167]. This was mediated by SIRT1 signalling and its downstream target, phosphorylated endothelial nitric oxide synthase (p-eNOS), which activated endothelial function and angiogenesis and reduced PTC loss. Upregulation of SIRT3/eNOS by BM-MSC-EVs also improved angiogenesis and regeneration in cisplatin-triggered AKI [108].

MFG-E8
Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) is a glycoprotein that inhibits the RhoA/ROCK signalling pathway. BM-MSC-EVs delivered MFG-E8 to rats with UUO and reduced inflammation, macrophage infiltration, mitochondrial damage, apoptosis, oxidative stress, and the EMT within two weeks [135].

Melatonin and PrP c
A recent study focused on the efficacy of melatonin in autologous MSC-based therapeutics for CKD [170]. Exposure of adipose-MSCs to melatonin upregulated expression of miR-4516 and cellular prion protein (PrP C ), and "MT exosomes" were harvested. Adipose-MSCs were also collected from patients with CKD (CKD-MSCs) and incubated with MT exosomes, which promoted proliferation, mitochondrial activity, and angiogenic proteins, and protected cells from senescence. These MT exosome-treated CKD-MSCs improved neovascularisation and functional recovery when administered to mice with hindlimb ischaemia, which was mediated through miR-4516-PrP c signalling.

Conclusions
MSCs have shown increasing potential in immunomodulation and regenerative medicine and their paracrine effects are mediated by the secretion of EVs [42,[181][182][183][184][185]. MSC-EVs are advantageous over their counterpart whole cells due to a higher safety profile, lower immunogenicity, and the inability to directly form tumours [42,[181][182][183][184][185]. The regenerative capacity of MSC-EVs is based on the cargo of biomolecules they deliver to injured renal cells, particularly the types of miRNA and ncRNA [60]. To minimise the level of reporting and publication bias in this review, multiple databases were searched, and two extensive tables were created to methodologically analyse 34 preclinical animal models of AKI and 26 of CKD. However, the heterogeneous nature of EVs means the extrapolated results are difficult to generalise. It can be concluded that MSC-EVs induce tubular proliferation, regeneration, and angiogenesis, and suppress apoptosis, oxidative stress, inflammation, the EMT, and tubulointerstitial fibrosis. By altering the pathogenesis of disease, MSC-EVs show promise in mitigating AKI and CKD and offering a novel therapeutic for patients.
There are some limitations of this review. There is no consensus regarding the reporting of studies using EVs as it is an emerging therapeutic and there is a lack of global standardisation in isolation, characterisation, and validation protocols [4,40,49]. Moreover, there are no established methods to differentiate the subtypes of EVs and therefore studies claiming to use a certain subtype cannot be verified and this makes comparison difficult. Additionally, there are functional differences in efficacy between EV subtypes. A recent study found adipose-MSC-MVs reduced proteinuria while only exosomes promoted natriuresis following chronic renal artery stenosis [147]. To minimise these effects, studies were compared based on the ISEV recommendations, according to the source of MSCs, EV subtype, protein content of administered EVs, and route of injection [49].
Furthermore, clinical translation is in its infancy and the conclusions are limited to preclinical animal models. They are monocausal and simplistic when compared to the multifactorial aetiologies of CKD and comorbidities, such as increasing age and cardiovascular disease, from which patients suffer [5]. Moreover, the selected animals are young and only a short duration of disease (weeks-months) is observed. Therefore, there is an increasing need for human clinical trials. In a phase II/III trial, twenty patients with Stage III and IV CKD (eGFR 15-60 mg/mL) received two doses of umbilical-MSC-EVs (100 µg/kg/dose) one week apart, and this increased eGFR and reduced serum creatinine, BUN, and urinary albumin creatinine ratio within one year [186]. The clinical improvement was attributed to increased anti-inflammatory cytokines and decreased TNF-α.
For translation of EV therapy to clinical practice, the following manufacturing issues surrounding optimal dosing, mode of injection, schedule of administration, potency assays, minimising dose toxicity, uniformity between batches, identification of EVs, and safety must be standardised [4,40,187]. This is inherently difficult when considering the heterogeneity of EVs, so each batch will display both donor and clone-specific differences [97,187]. Most studies used a single dose of EVs, but this may be insufficient to achieve a sustained effect in humans [113]. Multiple doses of EVs showed greater efficacy than single dosing but repeated injections decrease feasibility [98,109]. Most studies focused on intravenous injection but there is a shifting focus to delivering therapeutics to organs via their arterial blood supply. This maximises the efficacy at the target site while reducing its metabolism and systemic side effects [107]. The timing of EV injection is also significant whereby a recent study confirmed administration of BM-MSC-EVs after renal damage is more effective than delivering them prophylactically [154]. Most studies use in-house manufacturing and characterisation protocols to isolate EVs [97] and only a handful have published their adherence to good manufacturing practice criteria [187][188][189]. Further pharmaceutical regulation of the manufacture and delivery of EV-based therapeutics is required before they can be safely translated from the laboratory bench to the bedside [187].
In conclusion, MSC-EV therapy shows increasing potential for alleviating AKI and slowing the progression of CKD. Future studies should engineer the surface and cargo of EVs for superior specificity and develop optimal protocols for delivery and safe transition into clinical practice.

Conflicts of Interest:
The authors declare no conflict of interest.