Extracellular micro/nanovesicles rescue kidney from ischemia‐reperfusion injury

Abstract

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell‐to‐cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC‐derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC‐derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell‐free approach to treat renal IRI.

This review provides an overview of the current knowledge related to the potential use of extracellular vesicles (EVs) in protecting kidney from ischemia–reperfusion injury and discuss the promising future of EVs as an alternative to conventional cell therapy approaches.

1. INTRODUCTION

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming (Rodríguez et al., 2017). ARF is a syndrome characterized by a rapid decline in kidney function, resulting from a number of different causes (van Spil, Steenbergen, & Verhave, 2016; Yalavarthy, Edelstein, & Teitelbaum, 2007). Ischemia‐reperfusion injury (IRI) is the most common cause for this condition (Malek & Nematbakhsh, 2015). The term ischemia‐reperfusion describes a situation in which blood supply returns to the tissue after a period of insufficient perfusion (Sanderson, Reynolds, Kumar, Przyklenk, & Hüttemann, 2013). Reperfusion of ischemic tissue is associated with a wide and complex array of inflammatory responses that result in tissue damage through the induction of oxidative stress (Dorweiler et al., 2007). After blood flow to the ischemic kidney, oxidative damage mediated by reactive oxygen species (ROS) initiates a series of harmful cellular responses, leading to inflammation, apoptosis, kidney endothelial and tubular cell injuries, and finally acute renal dysfunction (Malek & Nematbakhsh, 2015). In particular, IRI is inevitable in solid organ transplantation and can determine the graft survival (Guibert et al., 2011). Although IRI‐associated injury can be decreased by keeping the organ in a cold temperature, it cannot be completely avoided (Vallant et al., 2015). Previous studies have proved that inflammatory cells, especially macrophages, T‐cells, and pro‐inflammatory cytokines, play an integral role in the initiation and extension of renal IRI (Wan et al., 2014; Zou et al., 2014). Inflammatory cascade induced by IRI exacerbates renal damage, so inhibition of inflammatory responses is a potential therapeutic modality to protect the kidney against IRI (Jansen et al., 2017; Nakamura et al., 2016). Mesenchymal stromal cells (MSCs) have the capability to alleviate many immunological disorders by their immunomodulatory properties (Klinker & Wei, 2015; Y. Wang, Chen, Cao, & Shi, 2014). In experimental models of kidney IRI, administration of MSCs improves kidney histological and functional recovery with no evidence of cell engraftment, signifying that repair process may be achieved by paracrine factors released by these cells (Shen et al., 2016). It is proposed that MSCs exert their therapeutic effects, at least in part, through the secretion of extracellular vesicles (EVs; De Jong, Van Balkom, Schiffelers, Bouten, & Verhaar, 2014). Preclinical studies demonstrate that EVs have a significant therapeutic potential as an alternative to whole cell transplantation (Di Rocco, Baldari, & Toietta, 2016). A superior safety profile compared with their parent cells and the ease of storage without losing function has added to their therapeutic appeal (Rani, Ryan, Griffin, & Ritter, 2015). This review provides an overview of the current knowledge related to the potential use of EVs in protecting kidney from IRI and discusses the promising future for EVs as an alternative for conventional cell therapy approaches.

2. GENERAL CHARACTERISTICS OF EXTRACELLULAR VESICLES; BIOGENESIS, COMPOSITION, AND MECHANISM OF ACTION

EVs were initially supposed to be cellular debris or a way to dispose unwanted molecules from cells, but an increasing body of evidence proved their crucial role in various physiological or pathological processes (Hood, San Roman, & Wickline, 2011; Yáñez‐Mó et al., 2015). They shuttle biological information between cells and represent a newly discovered mode of intercellular communication (Deddens, Vrijsen, Girao, Doevendans, & Sluijter, 2017). EVs also have some other physiological functions such as trafficking of membrane proteins and lipids, modulation of the immune system, senescence, angiogenesis, and cellular proliferation (Koniusz et al., 2016; Yáñez‐Mó et al., 2015). Based on their mechanism of release, three types of EVs have been classified. The vesicles in the first group, exosomes, are nanovesicles (50–90 nm) of endocytic origin with a density ranging from 1.09 to 1.18 g/ml, which are secreted through the fusion of multivesicular bodies (MVB) with the plasma membrane (Raposo & Stoorvogel, 2013). The second group, microvesicles (MVs), are released by direct budding from the plasma membrane through the dynamic redistribution of phospholipids. MVs shedding is associated with an elevated level of intracellular Ca²⁺ concentration and resulting activation of several key enzymes notably calpain, scramblase, and floppase (Camussi, Deregibus, Bruno, Cantaluppi, & Biancone, 2010; Lawson, Vicencio, Yellon, & Davidson, 2016; Tricarico, Clancy, & D'Souza‐Schorey, 2016). The third type of vesicles are referred to as apoptotic bodies, sized between 1,000 and 5,000 nm, released through fragmentation of plasma membrane that are produced during apoptosis (Bruno, Porta, & Bussolati, 2016). A schematic illustration of EVs production is shown in Figure 1. When referring to EVs, the majority of studies contain a heterogeneous population of exosomes and MVs, whereas the apoptotic bodies are different in acting and content (Greening, Xu, Gopal, Rai, & Simpson, 2017; Szatanek et al., 2017). However, due to limitations of isolation methods, apoptotic bodies and protein aggregates may also exist in the harvested samples (Livshits et al., 2016). In terms of composition, endosomal sorting complex required for transport machinery plays an important role in the clustering of cargo present in the EVs (Kwon, Oh, Nacke, Mostov, & Lipschutz, 2016; Leblanc et al., 2017). The presence of known cellular proteins in EVs has been analyzed extensively in previous studies (Koller, Patel, Kim, & Chen, 2017; Mizutani et al., 2017). Proteomic studies demonstrate a subset of cellular proteins that are targeted specifically to EVs. In addition to EV‐specific proteins, other ubiquitous cytoplasmic proteins may also exist in their cargo (Kalra, Drummen, & Mathivanan, 2016). They include cytoskeletal proteins such as tubulin, actin, and actin binding proteins. EVs also contain ANNEXINS, RABPROTEINS, signal transduction‐related proteins, heat shock proteins, tetraspanins, various metabolic enzymes, and MHC Class I/II molecules (Beach, Zhang, Ratajczak, & Kakar, 2014; Bosque et al., 2016; Kowal, Tkach, & Théry, 2014). EVs are also loaded with nucleic acid cargoes including mRNAs, microRNAs (miRNAs), and other noncoding RNAs (ncRNAs; Lötvall & Valadi, 2017). The discovery that EVs may elicit epigenetic modifications by transferring selected RNA molecules between cells has sparked a great interest in EVs (Lu et al., 2017). MicroRNAs are small ncRNAs that are involved in posttranscriptional gene silencing via binding to the 3′‐untranslated region or the open reading frame region of target mRNAs (Dinh, 2016; Makino et al., 2017). In addition to being packed in EVs, some miRNAs, known as circulating miRNAs, can stably exist in various biological fluids including blood, saliva, and breast milk (K. Wang, 2017; J. Wang, Chen, & Sen, 2016). With regard to lipid composition, they are almost identical to the lipid composition of the plasma membrane of producing cells. The enrichment of late endocytic related lipids has been reported in B cell derived exosomes. Phosphatidylserine also exists at the surface of exosomes isolated from dendritic cells and platelets (Skotland, Sandvig, & Llorente, 2017; Théry, Zitvogel, & Amigorena, 2002). Although limited data are available, these studies imply the sub‐cellular compartment origin of exosomes. Several mechanisms have been proposed for the EV‐target cell internalization. They can be taken up by the recipient cell through direct membrane fusion, clathrin‐mediated endocytosis, caveolin‐dependent internalization, macropinocytosis, and phagocytosis (Huang‐Doran, Zhang, & Vidal‐Puig, 2017; Mulcahy, Pink, & Carter, 2014; Prada & Meldolesi, 2016). The ligand–receptor interaction between transmembrane proteins on the EV membrane and receptors on the cell membrane leads to stimulation of signaling cascades in the target cell (Huang‐Doran et al., 2017; Rivoltini et al., 2016). Upon transfer, biological information within EVs can be exploited in the recipient cells. For example, m‐RNAs can be translated into proteins or micro‐RNAs can alter the gene expression profile of target cells (Hannafon & Ding, 2013).The fact that EVs contain multiple biomolecules endows them with huge potential for information transfer in comparison with known single‐molecule messengers, such as neurotransmitters, cytokines, growth factors, and other mediators (El Andaloussi, Mäger, Breakefield, & Wood, 2013; Lamichhane et al., 2014). And because EVs present membrane receptors that exist in the producing cell, they may convey the same changes in target cells. In this regard, it has been shown that the presence of MHC Class II molecules on exosomes isolated from antigen presenting cells can activate cognate T cells (Robbins & Morelli, 2014). The therapeutic efficacy and cargo content of EVs depend on the type and condition of the producing cell (Jarmalavičiūtė & Pivoriūnas, 2016). For example, the RNA content of EV derived from specific cell type in different culturing conditions is totally different, suggesting that EV biogenesis is a dynamic process adopting its message based on the cell's microenvironment (Ciardiello et al., 2016; Minciacchi, Freeman, & Di Vizio, 2015; Riazifar, Pone, Lötvall, & Zhao, 2017).

Figure 1.

Release of microvesicles, exosomes, and apoptotic bodies. Microvesicles are released through direct budding from the plasma membrane, whereas exosomes are produced as ILV by budding into EE and forming MVB. MVBs fuse with the plasma membrane and release their exosome cargo. Apoptotic bodies are produced by cells undergoing apoptosis. EE: early endosomes; ILV: intra luminal vesicles; MVB: multi vesicular bodies [Color figure can be viewed at wileyonlinelibrary.com]

3. REGENERATIVE POTENTIAL OF STEM CELL‐DERIVED EVs

Regenerative medicine seeks to restore a damaged, malfunctioning, or defective tissue (Gonzalez‐Garza & Cruz‐Vega, 2017). The major drawbacks associated with organ transplantation, such as great demand and unmet supply, immune rejection, and the required chronic immunosuppression treatment have driven efforts to develop alternative approaches. Rouchi and Mahdavi‐Mazdeh (2015). Cell therapy is one of the most promising strategies in regenerative medicine (Napoli, Lippert, & Borlongan, 2018). In conventional cell therapies, cell populations are isolated, expanded in vitro, and administered at the site of defect or systematically, hoping that injected cells may home in on the injured area (Burke et al., 2016). Because of the paucity of stem cells, reaching a clinically sufficient number of cells in vitro expansion is inevitable (Javazon, Beggs, & Flake, 2004; Marks, Witten, & Califf, 2017). The current culturing systems are challenged by the adverse effects of dedifferentiation and induction of senescence, which may affect a cell's therapeutic function (Musacchio & Veronese, 2017; Turinetto, Vitale, & Giachino, 2016). Furthermore, in the context of cell therapy, intravenously administered cells may undergo lung and other vital organs entrapment (Fischer et al., 2009). Long‐term survival of transplanted cells and unwanted differentiation in vivo are other pitfalls of conventional cell‐based therapy approaches (Lee, Choi, Cha, & Hwang, 2015). Therefore, at least with regard to safety profile, cell free approaches are superior to conventional cell‐based therapies (Raik, Kumar, & Bhattacharyya, 2017; Toh, Lai, Hui, & Lim, 2017). Recent studies on stem cell‐based therapies have shed light on the mechanism by which stem cells exert their protective effects. Research shows that structural contribution of administered cells in the restored tissue is limited and that rather the paracrine secretion plays the major role (Lee, El Andaloussi, & Wood, 2012; Rosca, Rayia, & Tutuianu, 2017; Trohatou & Roubelakis, 2017). This hypothesis has sparked interests in paracrine secretions including growth factors, cytokines, and EVs (Asai et al., 2017; Ong et al., 2017; Trohatou & Roubelakis, 2017). The therapeutic effects of EVs acquired from diverse sources of stem/progenitor cells on tissue regeneration have been well documented in previous publications (Nishida‐Aoki et al., 2017; Riazifar et al., 2017; Varkouhi et al., 2017). From the therapeutic point of view, stem cell‐isolated EVs have been used for their ability to improve different cellular processes, such as proliferation, angiogenesis, and immune modulation (Hofer & Tuan, 2016; Nooshabadi et al., 2018). EVs fall in the intermediate range of current therapeutic modalities for regenerative medicine (Abreu, Weiss, & Rocco, 2016). As they are a package of different proteins and multiple types of coding and noncoding nucleic acids, the function they exhibit or the response they elicit may be more efficient and longer‐lasting compared with those induced by individual biomolecules (Bruno et al., 2017; Jay & Vunjak‐Novakovic, 2017; Lo Sicco et al., 2017). They have the ability to circumvent organ entrapment and cross other physiological barriers (El andaloussi, Lakhal, Mäger, & Wood, 2013; Wood, O’Loughlin, & Lakhal, 2011). EVs can also be engineered to possess desired properties and can be enriched with reagents of interest. They can be stored without considerable function loss and they do not run the risks associated with cell maldifferentiation (Candelario & Steindler, 2014; Katsuda, Kosaka, Takeshita, & Ochiya, 2013; Nawaz et al., 2015). The regenerative potential of stem cell‐derived EVs in several models of tissue injury is listed in Table 1.

Table 1.

Published studies on EVs regenerative potential in various ischemic tissue injury models

Target tissue/model	Species exosome	Cell type	Method	Dose	Ref
Murine hepatic ischemia/reperfusion injury (IRI) Mice	Mouse	Bone marrow–derived mesenchymal stem cells	Ultracentrifugation	2 × 1010 particles/body	Haga et al. (2017)
Hindlimb ischemia mouse model	Mouse	Mesenchymal stem cells (MSCs)	Ultracentrifugation	100 μg	Gangadaran et al. (2017)
Mouse model of myocardial ischemia/reperfusion injury	Human	ESC‐derived mesenchymal stem cell	Ultracentrifugation	3.5 µg/ml	Lai et al. (2010)
Myocardial infarction rats	Human	Cardiosphere‐derived cells	Ultracentrifugation	10 µg	Cambier et al. (2017)
Acute hind limb ischemia	Human	Serum‐derived from healthy blood donors	Ultracentrifugation	2 × 1011 particles	Cavallari et al. (2017)
Lung ischemia‐reperfusion (IR) injury (C57BL/6 wild‐type [WT] mice)	Human	Mesenchymal stromal cells	Ultracentrifugation	3 × 106 particles	Stone et al. (2017)
Hypoxic rat liver perfusion model	Human	Human liver stem‐like cells (HLSC)	Ultracentrifugation	100 μg	Rigo et al. (2018)
Protect SH‐SY5Y nerve cells against ischemia/reperfusion injury	Human	Endothelial cell‐derived	Ultracentrifugation	5 μg	Xiao et al. (2017)
Acute myocardial infarction in mice	Human	Plasma	Differential centrifugation	approximately 3 × 108 EV/ml to 5 × 108 EV/ml	Akbar et al. (2017)
Liver ischemia reperfusion injury	Mice	Dendritic cells	Differential centrifugation	1 × 106	Zheng et al. (2018)
Swine model of chronic ischemia	Human	Mesenchymal stem cells	Ultracentrifugation	lg extracellular vesicles in 2 ml of 0.9% saline	Potz et al. (2018)
Myocardial infarction	Murine	Induced pluripotent stem cell (iPSC)	Ultracentrifugation	100 µg	Adamiak et al. (2018)
Ischemic/reperfusion liver injury	Rat	Bone marrow mesenchymal stromal cell	Ultracentrifugation	270 μg/ml	Damania, Jaiman, Teotia, and Kumar (2018)
Hepatic ischemia‐reperfusion (I/R) injury	Human	Human‐induced pluripotent stem cell–derived mesenchymal stromal cells	Differential centrifugation	2.5 × 1012 particles	Du et al. (2017)
Myocardial ischaemia/reperfusion injury	Rat	Plasma	ExoQuick precipitation solution	10 µg	Minghua et al. (2018)

4. APPLICATION OF STEM CELL–DERIVED EVs TO ALLEVIATE KIDNEY IRI

When the injury is severe, incomplete renal recovery may lead to the proliferation of fibroblasts and excessive deposition of extracellular matrix proteins. Therefore the patients who have had renal IRI are at high risk for the development of end‐stage renal disease (Bellomo, Kellum, & Ronco, 2012; Farooqui, Pommergaard, & Rasmussen, 2017; Zhang et al., 2018). Studies show that prevention of inflammatory reaction and oxidative stress is a good choice for protecting organs from acute IRI (Gong et al., 2014; Malek & Nematbakhsh, 2015). Research has reported that MSCs and their EVs can reduce inflammation and attenuate IRI‐induced organ dysfunction (DeSantiago, Bare, & Banach, 2013; Hu & Zou, 2017). In this regard, Lin et al. tested the hypothesis that a combined use of adipose tissue mesenchymal stromal cells (ADMSC) and ADMSC‐derived exosomes can protect rat kidney from acute IRI. To test this view, adult‐male SD rats (n = 40) were randomly divided into Group 1 (sham control), Group 2(IRI), Group 3 (IRI + exosome), Group 4 (IRI + ADMSC), and Group 5 (IRI + exosome + ADMSC). After 72 hr, the creatinine level and kidney injury score were measured to be the lowest in Group 1 and the highest in Group 2, significantly higher in Group 3 than in groups 4 and 5, and significantly higher in Group 4 than in Group 5. The protein expression of inflammatory, oxidative‐stress, apoptotic, and fibrotic biomarkers showed the same pattern, whereas the antiapoptotic and angiogenesis biomarkers showed an opposite pattern of creatinine level in all groups. Finally, they concluded that combined exosome–ADMSC therapy was superior to either one for protecting the kidney from acute IRI (Lin et al., 2016). Kalpinen et al. investigated the effects of IFN‐ɣ stimulus on the secretion and function of EVs originated from umbilical cord blood‐derived MSCs. Cells were grown in serum free media with or without IFN‐ɣ incubation and the EVs were harvested by ultracentrifugation. The protein composition of the produced EVs was analyzed with regard to their therapeutic effect in renal ischemia‐induced injury in an in vivo model. They found that only EVs derived from cells with no inflammatory stimulus can protect rat kidneys from IRI. IFN‐ɣ stimulation markedly changed EV protein content (Kilpinen et al., 2013). In line with previous studies, to explore the potential mechanism of protective effects of MSC‐exosomes on the kidney, Shen et al. reported that C‐C motif chemokine receptor‐2 (CCR2) plays a major role in therapeutic effects of MSC‐derived exosomes. They could prove that high expression of CCR2 on MSC‐exosomes can reduce the level of its ligand (CCL2) and suppress its effects to attract immune cells or activate them. They also found that knock down of CCR2 greatly abolished the protective effects of MSC‐exosomes against kidney IRI. This study indicates that CCR2 is a key molecule on MSC‐exosomes, which regulates inflammation and acts as a decoy for CCL2 suppression (Shen et al., 2016). Gatti et al. isolated MVs from MSCs and injected intravenously in a rat model of ischemic kidney injury 45 min after the ischemia induction. They found that a single injection of MVs protects rats from acute kidney injury by preventing apoptosis and inducing tubular epithelial cell proliferation, which significantly improved renal function compared with the control group. Moreover, MVs inhibited the progression of acute injury into subsequent chronic kidney disease. The protective effects of MVs were abrogated following treatment with RNase, implying that these effects are related to RNA cargo of MVs (Gatti et al., 2011). Zou et al. studied the therapeutic effects of human Wharton‐Jelly MSC‐ derived MVs (hWJMSC‐MVs) on renal IRI and its potential mechanisms. To do this, MVs were harvested from conditioned medium and systematically injected in rats immediately after ischemia of the left kidney for one hour. The results showed that in the groups treated with hWJMSC‐MVs, renal epithelial cells apoptosis was reduced and proliferation was improved. IRI significantly enhanced the expression of CX3CL1 in the kidney. Whilst, MVs treatment downregulated its expression. By binding to its only known receptor (CX3CR1), CX3CL1 aids in the infiltration of macrophages to the inflammation site. In this study, they could show that downregulation of CX3CL1 by MVs might be a way to decrease the recruitment of macrophages in the kidney and MVs’ immune modulatory properties may be one of the underlying mechanisms of hWJMSC‐MVs’ protective effects against renal IRI (Zou et al., 2014). Vinas et al. reported that transplantation of endothelial colony forming cells (ECFCs) or their EVs can protect kidney against IRI. They studied the miRNA profile of isolated EVs and their role in renal and endothelial cell protection. ECFC‐isolated EVs were transfected with miRNA‐486‐5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. Endothelial cells were subjected to hypoxia and then were incubated with ECFC‐EVs. It was observed that the miRNA‐486‐5p content was increased and the AKT phosphorylation pathway was activated, whereas PTEN was diminished. MiRNA 486‐5p targets PTEN, which in turn inhibits apoptosis. In mice with renal IRI, administration of ECFC‐EVs showed a strong protective function. In addition, the miRNA‐486‐5p levels in injured kidney were elevated and PTEN was decreased. Therefore, infusion of ECFC‐EVs ameliorates renal IRI via transfer of miRNA‐486‐5p targeting PTEN (Viñas et al., 2016). An accumulating body of evidence has suggested that mitochondrial dynamics play an important role in IRI (Calo, Dong, Kumar, Przyklenk, & Sanderson, 2013). Mitochondria are a class of dynamic subcellular entities that constantly experience fission and fusion. Under stressful situations, the mitochondrial dynamics are changed to the fission state, leading to mitochondrial fragmentation and cell death (Murphy et al., 2016). Approaches that inhibit mitochondrial fragmentation result in renal protection against injury (Liesa, Palacín, & Zorzano, 2009; Zhan, Brooks, Liu, Sun, & Dong, 2013). Given that miRNA‐30 has been reported to regulate mitochondrial fragmentation, Gu et al. hypothesized that WJMSCs‐isolated EVs may regulate mitochondrial fission via miR‐30, thereby inhibiting renal IRI. They isolated the EVs from the medium of MCSs via ultracentrifugation method and injected intravenously in rat promptly after AKI induction. The animals were killed after one day of reperfusion and the samples were harvested for histopathological and molecular studies. The morphology of the mitochondria was observed by Mito tracker Red staining. The expression of dynamine related protein 1 (DRP1) was estimated by western blot. The apoptosis pathway was identified by immunostaining and the level of miR‐30 in EVs was assessed by qRT‐PCR. The results showed that renal injury significantly reduced miR‐30 expression and turned the mitochondrial dynamics into fission state. In the group treated with EVs, both changes were restored along with reduced cell apoptosis. What is more, when EVs were incubated with miR‐30 antagomirs and their protective effect was substantially decreased. Thereby, the miR‐30 cargo of WJMSCs‐EVs can inhibit mitochondrial fission and protect kidney from IRI (Gu et al., 2016). Several research works have proved the contribution of the glomeruli specific MSCs (Gl‐MSCs) in kidney repair after injury. These cells express CD133, CD24, and embryonic renal marker PAX2 (Bussolati et al., 2005). Ranghino et al. tested the hypothesis whether Gl‐MSCs and their EVs can protect kidney against IRI. They also compared the effects of Gl‐MSCs and Gl‐MSCs‐derived EVs with the therapeutic function of CD133+progenitor cells harvested from the human tubule of the renal cortical tissue (T‐CD133+) and their EVs (T‐CD133+‐EVs). Renal IRI was created in SCID mice for 35 min. Then., the animals were divided in different groups that were treated with: GI‐MSCs, T‐CD133+cells, GI‐MSCs‐EVs, and T‐CD133+‐EVs. To check the renal function, plasma creatinine and blood urea nitrogen levels were measured on the second day after IRI. The results showed that both Gl‐MSCs and their EVs could enhance tubular epithelial cell proliferation and renal function. In addition, T‐CD133+cells, but not their EVs, could aid in renal recovery compared with the control group. EVs treated with RNase were ineffective in ameliorating renal function (Ranghino et al., 2017). It has been shown that administration of adult renal cells can be a successful therapy to ameliorate kidney IRI (Kelly, Zhang, Wang, Zhang, & Dominguez, 2012). Dominguez et al. could show that EVs isolated from adult rat renal tubular cells significantly enhanced kidney function when injected intravenously 24 and 48 hr after ischemia induction. Furthermore, they observed that EV therapy significantly ameliorated injuries to renal tubular cells, triggered 4‐hydroxynanoneal adduct formation, modulated inflammatory responses, prevented fibrosis, and improved microvascular pruning. In the groups treated with EVs, there was also a substantial reduction in renal transcriptome drift, which was observed after IRI (Dominguez et al., 2017). Wu et al. used MVs derived from WJMSCs on kidney IRI after cardiac arrest renal transplantation in rats. MVs were administered intravenously in rats promptly after kidney transplantation. The animals were killed at 24 hr, 48 hr, 1 and 2 weeks after transplantation. The levels of von Willebrand Factor (vWF), tumore necrosis factor (TNF)‐α, and interleukin (IL)‐10 were evaluated using ELISA. The proliferation and apoptosis of tubular cells were determined using Ki67 immunostaining and TUNEL assay, respectively. Masson's tri‐chrome straining and alpha‐smooth muscle actin (α‐SMA) staining were used to determine the extent of renal fibrosis. The recruitment of macrophages was assessed via CD68+staining. The results showed that in the animals treated with WJMSCs‐derived MVs, there was a significant enhancement of survival rate and renal function. In addition, WJMSCs‐MVs group renal cells death was significantly reduced and MVs could improve their proliferation. MVs could also alleviate inflammation and renal fibrosis (Wu et al., 2018). Schematic illustration of potential mechanisms by which MSC‐derived EVs exert their protective effects against renal IRI are shown in Figure 2.

Figure 2.

Upon ischemia reperfusion injury, inflammatory cells produce different types of chemokines and ROS that leads to oxidative stress and tubular cells apoptosis. EVs mediate tubular epithelium recovery by enhancing angiogenesis, reducing oxidative stress/tubular cells apoptosis, and modulating inflammatory responses. MiRNA‐486‐5p cargo of EVs stimulates AKT signaling pathway and mitigates PTEN pathway which results in cell survival and reduced apoptosis respectively. MiRNA‐30 in EVs inhibits mitochondrion fission and protects renal tubular cells against IRI. Downregulation of CXCL‐1 by EVs reduce the infiltration of macrophages and modulate inflammatory responses. EVs: extracellular vesicles; IRI: ischemia–reperfusion injury; ROS: reactive oxygen species [Color figure can be viewed at wileyonlinelibrary.com]

5. CONCLUSION AND PERSPECTIVES

Renal IRI represents a worldwide clinical challenge of increasing incidence, which is associated with considerable morbidity and mortality. In the absence of efficient therapeutic modalities, the treatment of patients suffering from renal IRI mostly relies on supportive maneuvers. Stem cell administration at the time of renal IRI and/or at later times may ameliorate kidney function and accelerate regeneration process. EVs are emerging therapeutics which mediate stem cell responses during tissue homeostasis and its restoration after injury. Because EVs provide an efficient route for cell‐cell communication, EVs offer several benefits as compared with producing cells. Stem cell‐ derived EVs can be modified to have desired biological function, and they can possess exceptional biocompatibility obviating the risks associated with total cell infusion. Their small size endows them to avoid the pulmonary first‐pass effect and to penetrate deep inside most body barriers. Preclinical studies of EV therapy in renal IRI shows promising results and opens new avenues for the management of this condition.

CONFLICTS OF INTEREST

The authors declare that no financial or other potential competing interests exist with regard to this study.