Stem Cell-Derived Extracellular Vesicles and Kidney Regeneration

Extracellular vesicles (EVs) are membranous vesicles containing active proteins, lipids, and different types of genetic material such as miRNAs, mRNAs, and DNAs related to the characteristics of the originating cell. They possess a distinctive capacity to communicate over long distances. EVs have been involved in the modulation of several pathophysiological conditions and, more importantly, stem cell-derived EVs appear as a new promising therapeutic option. In fact, several reports provide convincing evidence of the regenerative potential of EVs released by stem cells and, in particular, mesenchymal stromal cells (MSCs) in different kidney injury models. Described mechanisms involve the reprogramming of injured cells, cell proliferation and angiogenesis, and inhibition of cell apoptosis and inflammation. Besides, the therapeutic use of MSC-EVs in clinical trials is under investigation. This review will focus on MSC-EV applications in preclinical models of acute and chronic renal damage including recent data on their use in kidney transplant conditioning. Moreover, ongoing clinical trials are described. Finally, new strategies to broaden and enhance EV therapeutic efficacy by engineering are discussed.


Introduction
Renal failure is one of the most significant causes of mortality and morbidity all over the world [1]. Acute kidney injury (AKI) is a major clinical problem, affecting up to 5% of all hospitalized patients with acute illness, thus having a great impact on public health resources [2]. AKI is traditionally defined by a rapid decline of renal function, which clinically manifests as an increase of urea and creatinine in serum, associated with disruption of salt and water homeostasis. More importantly, about 8% to 16% of patients with AKI progress to chronic renal failure [3]. There is evidence that even a single episode of AKI predisposes the kidney to maladaptive response to injury leading to progressive loss of function and the development of chronic kidney disease (CKD) [4,5]. In parallel, the incidence of CKD has increased, mainly due to the enhanced prevalence of diabetes and obesity [6]. The current therapies for CKD concentrate on slowing disease progression and, despite beneficial effects, are not sufficient to counteract the disease evolution. A large proportion of patients with end-stage renal disease undergo hemodialysis and/or renal replacement therapy, the latter option with high costs and significant limitation in organ availability [7,8]. Finding new therapeutic strategies for AKI and CKD remains an ongoing quest. In the last decades, innovative stem cell therapies have been tested both as preclinical development and in pilot clinical trials, demonstrating the efficacy of these novel approaches [1,5,9]. More recently, extracellular vesicles (EVs), bioproducts released physiologically

MSC-EVs and Tissue Regeneration
The growing evidence in Regenerative Medicine supports the hypothesis that stem cells exert their therapeutic effect by a paracrine/endocrine manner rather than a direct repopulation of the injured tissues [39][40][41][42]. This postulate was strongly supported by numerous in vivo studies demonstrating that the therapeutic benefit of stem cells is orchestrated by their secretome, composed by growth factors, cytokines, chemokines, and EVs [13]. In particular, regarding renal regeneration, Bi et al. [43] showed that the injection of conditioned media from MSCs limits apoptosis and enhances proliferation of tubular cells after a toxic injury, thus promoting kidney repair. The use of EVs, and in particular stem cell-derived EVs, has been proposed as an alternative to stem cell therapy for the regeneration of several injured organs [9,41,44]. MSC-EVs may be isolated from MSCs of different adult tissues such as bone marrow, adipose tissue, peripheral blood, and neonatal birth-associated tissues including placenta, umbilical cord, and cord blood [45]. They are characterized by the expression of the typical mesenchymal stromal markers which include CD44, CD73, CD90, CD105, and CD146 [46].
Moreover, the use of EVs presents many advantages compared with their originating cells, like higher safety profile, lower immunogenicity, and the unfeasibility to maldifferentiate [47][48][49]. They display excellent biological tolerance, an important requirement for therapeutic applications [50]. In addition, EVs possess unique targeting and delivering features as they may be rapidly internalized into target cells [51].

MSC-EVs and Acute Kidney Injury
The regenerative capacity of EVs is sustained by a high number of publications, and several pre-clinical studies demonstrate that stem cell-derived EVs promote tissue repair and reduce inflammation in different AKI models (Table 1) [52]. The hallmark of AKI is the rapid reduction of renal function in parallel with tubular cell loss, resulting in increased blood urea nitrogen (BUN) and plasma creatinine [53]. In 2009, Bruno et al. [54] demonstrated that the effect of bone marrow (BM) MSC-EVs was superimposable to the one of the originating cells in a model of AKI induced by glycerol injection. BM MSC-EVs accelerate the recovery of injured tubular cells, promoting cell proliferation and protecting cells from apoptosis ( Figure 1) [9]. Since that work, many studies have been conducted to confirm the beneficial effect of EVs in several AKI models, and related mechanisms have been explored. At present, it is well recognized that EV activity mainly involves the horizontal transfer of genetic materials [54][55][56][57]. BM MSC-EVs carry specific mRNAs that, in turn, stimulate recipient injured cells for re-entry into the cell cycle [54]. Another group demonstrated that the transfer of human IGF-1 receptor mRNA, present in BM MSC-EVs, to tubular cells is fundamental to trigger renal recovery [58].
Moreover, it has been demonstrated that Drosha-knockdown MSCs release ineffective EVs, tested in in vivo AKI model, sustaining a central role of miRNA cargo [57]. MSC-EVs isolated from bone marrow cells were also tested in toxic AKI models, induced by cisplatin and gentamycin [59,60]. In all toxic models, BM MSC-EVs ameliorated renal function and reduced the classical histological lesions of the disease [4]. The same EV source resulted in an effective ischemia/reperfusion injury (IRI) model that mimics hypoxic insult, a common feature during AKI [61,62]. The effect of MSC-EVs isolated from other tissues was also tested in several AKI models. Similar positive results were obtained using cord blood MSC-EVs that promoted tubular cells dedifferentiation and growth, and Warton Jelly MSC-EVs, that stimulated proliferation and reduced inflammation and apoptosis via mitochondrial protection [63][64][65][66] (Figure 1). In addition, EVs obtained from glomerular MSCs and liver MSCs, human liver stem cells (HLSCs), resulted in protection from AKI [67][68][69]. Altogether these data indicate that MSC-EVs, isolated from different sources, are effective in the amelioration of preclinical models of AKI, targeting multiple aspects of the disease, stimulating cell proliferation, and reducing apoptosis, inflammation, and oxidation [4,9] (Figure 1).

Conditioning of the Kidney Transplant
Renal transplantation is significantly improving the quality of life of patients with end-stage renal disease; however, chronic allograft nephropathy limits the organ survival and more than one transplant might be required during patient life. The uses of MSCs and MSC-EVs are tested in various clinical protocols related to transplantation, to favor tolerance and to prolong allograft survival [70]. The preconditioning of a kidney with MSCs and MSC-EVs may be another interesting option to limit tissue damage due to ischemia-reperfusion injury and chronic allograft nephropathy. MSCs and MSC-EVs were tested in a rat model of kidney donation after cardiac death (DCD). DCD kidneys treated with MSC-EVs during organ cold perfusion (4 h), showed significantly lower signs of renal damage [71]. In addition, treated kidneys increased energy consumption with up-regulation of enzymes involved in energy metabolism [71]. This approach is gaining increased interest for the pre-transplant graft perfusion in several organs, as it appears to be able to abrogate or strongly reduce ischemic injury.

MSC-EVs and Chronic Kidney Disease
Several preclinical models are available to mimic the broad range of pathologies defined as CKD. The severity of CKD can manifest itself over time depending on numerous causes. One of the trigger causes is diabetes [72]. Hyperglycemia induces a cascade of events resulting in glomerular and tubule-interstitial fibrosis, with podocyte damage/loss and mesangial cell hypertrophy, a hallmark of diabetic nephropathy. The progression of fibrosis is the leading cause of renal dysfunction not only for diabetic nephropathy but also for other CKDs [73,74]. In this scenario, several groups tested in animal models of CKD, different doses, number, and timing of EV administration, with the intent to set optimal EV regimen ( Table 1). EVs isolated from urinary MSCs have been described as effective in the prevention of CKD progression by inhibiting apoptosis in a rat model of diabetic nephropathy induced by streptozotocin injection [75]. EVs induce a reduction of urine volume and apoptosis of podocyte and tubular epithelial cells (Figure 1). Urinary MSC-EVs carry transforming growth factor-β1, angiogenin, and bone morphogenetic protein-7, drivers of the observed reno-protective activity [75]. In addition, the direct administration of MSC exosomes under the renal capsule generated a rapid improvement of renal morphology, demonstrated in the same animal model [76]. Recently, EVs isolated from BM MSCs and from liver MSCs have been shown to be effective in the reversion of renal fibrosis in an already established diabetic nephropathy model [8]. MSC-EVs and HLSC-EVs contain a selection of antifibrotic miRNAs able to downregulate profibrotic genes, restoring normal renal function [8]. Similar positive results were obtained by multiple injections of HLSC-EVs in a CKD model induced by aristolochic acid [77].
Other in vivo models of CKD are the surgical five-sixth resection of the kidney tissue and the obstruction of the ureter, leading to glomerulosclerosis and fibrosis [78]. In both CKD models, multiple injections of BM MSC-EVs prevented renal failure [79,80]. In a similar model, combined with diet, multiple administrations of a conditioned medium, purified from human embryonic MSCs, slowed the deterioration of renal function [81]. Moreover, in a porcine model of metabolic syndrome and renal artery stenosis, a single intrarenal administration of adipose tissue-derived MSC-EVs reduced renal inflammation and fibrosis by delivery of IL10 [82].
The robustness of preclinical data about the therapeutic efficacy of MSC-EVs in acute and chronic models is encouraging to go further towards clinical studies.

MSC-EVs and Clinical Trials
The translation of EV-based therapy into clinical practice requires the clarification of several critical issues [13]. The major one to be considered is the identification of optimal protocols for EV production, isolation, and storage [13]. Similarly, the determination of potency assays to test the efficacy of each EV batch is mandatory. In fact, the majority of approved clinical trials implying EVs (listed in www.clinicaltrials.gov) focus on diagnostic purposes. However, at present, there are four clinical trials involving MSC-EVs for therapeutic use ( Table 2). Two of them are designed by Nassar et al. [83] at the Sahel Teaching Hospital of the University of Cairo. Both trials imply the use of EVs isolated from cord blood MSCs [83]. The first study aims to evaluate the effect of consecutive doses of MSC-EVs in 20 patients with type 1 diabetes, with a follow up of three months [13]. The results are not available yet. The second study enrolled 20 patients with CKD and results are already published [83]. The authors observed an improvement of renal function with amelioration of glomerular filtration, proteinuria, and BUN in patients one year after EV administration (two doses). Moreover, EVs displayed an anti-inflammatory activity, decreasing TNF-α and increasing IL-10. The results of this clinical study are promising in terms of feasibility and efficacy for MSC-EV therapeutic use. Another potential application in which preclinical studies are robust and convincing is the use of MSC-EVs to promote macular regeneration. There is an ongoing clinical trial in China focusing on the safety and efficacy of exosomes isolated from cord tissue-derived MSCs in patients with refractory macular holes in the eye. Finally, a clinical trial, which involves the injection of MSC-EVs engineered with miR-124 for the treatment of patients after acute ischemic stroke, was approved in Iran, The number of clinical trials on EVs as a therapeutic strategy will increase enormously in the next years and, hopefully, their use will enter into clinical practice.

EV Engineering and Future Strategies
In the constant quest to broaden the therapeutic applications of EVs, further approaches focused on the enhancement of EV efficacy by engineering. The natural origin of EVs, along with their spheroid shape and cargo ability, makes them ideal candidates for the efficient loading of therapeutic molecules [84]. The strategy to engineer EVs with pro-regenerative molecules or specific drugs is currently gaining an increasing interest [85]. EVs may be engineered to potentiate their therapeutic cargo by increasing the levels of active molecules (proteins or RNAs) already present within EVs or to modify their biodistribution/stability by changing the composition of surface molecules. The strategy to deliver therapeutic RNAs possesses an excellent potential and a wide range of applicability; however, the polar RNA molecules are exposed to rapid digestion by extracellular RNases [50,86]. The use of synthetic nanoparticles has also been explored with some limitations [87,88]. For these reasons, EVs are the central point of intense research [50]. At present, the existing methods for EV engineering are divided into two categories: Direct and indirect methods, indicating the direct modification of the EVs or the engineering of the cell of origin used for EV production.
Direct EV engineering can be done with multiple techniques: Incubation, electroporation, sonication, freeze/thaw cycles, and saponin-assisted method without significantly impairing EV constitution and functionality ( Figure 2) [89]. The incubation is a passive method preferred for the loading of hydrophobic compounds, with a higher efficiency compared to those obtained with liposomes. The reason may be the presence of particular domains within EV membranes, absent in artificial membranes of liposomes [90]. Sun et al. [91], for example, mixed purified EVs with curcumin, a natural compound with antioxidant and anti-inflammatory activities, and they demonstrated an increased efficacy compared with those of naive EVs when injected into mice with septic shock.
Exogenous genetic material (small RNAs or miRNAs) is generally added to EVs using electroporation, as they are hydrophilic molecules. To define the most efficient protocol, Pomatto et al. [92] tested different voltages and number of pulses and described 750 V and 10 pulses as the optimal one, with the highest RNA loading without significant EV damage [92].
As an example, a non-coding RNA (Lnc-RNA-H19) has been transfected into high-yield nano-EVs to create an effective drug delivery system for wound healing in diabetics [93]. In addition, sonication or cycles of a deep freeze and then slow thaw are two alternative methods for inserting different molecules into isolated EVs [94,95]. Moreover, it has also been shown that the saponin-assisted encapsulation method allows the highest loading efficacy and protection versus protease degradation [89]. All options mentioned above can be combined to improve final loading efficacy. The modification of the genome by CRISPR/Cas technology, which alone has low efficacy of delivery, is a new potential tool to be inserted into EVs by electroporation [96].
The second category of engineering technology is based on the modification of EV originating cells that allows subsequent isolation of EVs, which already express the desired molecule. For example, it has been demonstrated in an in vivo model of unilateral ureteral obstruction that MSCs engineered to overexpress miRlet7c selectively localize into the injured kidney and upregulate miR-let7c expression, attenuating kidney injury. Similarly, exosomes derived from engineered MSCs were able to selectively transfer miR-let7c to damaged kidney cells resulting in antifibrotic functions [97]. In a similar approach, MSCs were engineered to overexpress pro-regenerative miRNAs, such miR10a, miR127, and miR486, and deriving EVs were tested in models of acute renal injury [98]. EVs obtained from engineered MSCs were more effective than EVs derived from naïve MSCs when used at low doses [98].

Conclusions
The number of studies on the use of EVs, especially those derived from MSCs, for the treatment of AKI and CKD is continuously increasing, and EVs are considered a promising approach for tissue regeneration. The pro-regenerative effect of EVs is now well established for AKI, sustained by convincing results in a large number of different experimental models. The regenerating role of MSC-EVs in the slowdown of CKD, at variance, is still limited to a restricted number of preclinical models and should be better investigated. The translation of this approach for clinical use, based on ongoing and future clinical trials, will open a new scenario in Regenerative Medicine. Finally, EVs could be further exploited as a carrier for the delivery of exogenous materials such as RNAs, proteins or existing small drugs. An accurate setting of therapeutic doses and schedule are still needed.