Regenerative abilities of mesenchymal stem cells via acting as an ideal vehicle for subcellular component delivery in acute kidney injury

Abstract Cell‐to‐cell communication and information exchange is one of the most important events in multiple physiological processes, including multicellular organism development, cellular function regulation, external stress response, homeostasis maintenance and tissue regeneration. New findings support the concept that subcellular component delivery may account for the beneficial effects of mesenchymal stem cell (MSC)‐based therapy‐mediated protection against acute kidney injury (AKI). Through the secretion of extracellular vesicles (EVs), formation of tunnelling nanotubes (TNTs) and development of cellular fusions, a broad range of subcellular components, including proteins, nucleic acids (mRNA and miRNA) or even organelles can be transferred from MSCs into injured renal cells, significantly promoting cell survival, favouring tissue repair and accelerating renal recovery. In this review, we outline an extensive and detailed description of the regenerative consequences of subcellular component delivery from MSCs into injured renal cells during AKI, by which the potential mechanism underlying MSC‐based therapies against AKI can be elucidated.

fibroblast-like, multipotent progenitor cells that can be easily isolated from various adult tissues, including bone marrow, adipose tissue and the umbilical cord; characteristically, they are capable of differentiation, regeneration and immunomodulation. 8 It has been confirmed in several different experimental AKI models that the administration of MSCs can significantly improve kidney histologic and functional recovery. [9][10][11] It has also been shown that the apoptosis of renal tubular epithelial cells (RTECs) is common during AKI. Whereas the administration of MSCs displays a significant renoprotective effect by diminishing RTEC apoptosis. 12 Meanwhile, Zhang et al found that MSCs could accelerate the proliferation of endothelial cells, promoting angiogenesis and preventing microvascular dropout. 11 In terms of inflammatory cells, MSCs have the ability to reduce the infiltration of both neutrophils and macrophages, 13 decrease the proliferative and cytotoxic activity of NK cells, 14 suppress the maturation and differentiation of dendritic cells, 15 regulate T and B cells, 16,17 and turn macrophages from a pro-inflammatory phenotype M1 to an anti-inflammatory phenotype M2. 18 By interacting with various types of injured cells during AKI, MSCs showed that they have a role in minimizing injury, promoting regeneration, eliciting an immunological balance and, finally, contributing to the alleviation of renal injury. However, how these beneficial signals are transferred is still not well clarified.
Mainly, two hypotheses prevailed over the past decades related to the major repair mechanism underlying the protective effects of MSC-based therapies for AKI ( Figure 1). The first one suggested that injected MSCs migrated into the injured kidney, where they proliferated, engrafted and differentiated into normal kidney cells, promoting renal recovery. The second one proposed that the paracrine/endocrine activity of MSCs was responsible for their regenerative effects. However, the realization that the transient presence of MSCs within the injured kidney could neither account for cell differentiation nor for cell replacement led to an acceptance of the paracrine/endocrine-dependent mechanism by most investigators. 19 Commonly, it was thought that MSCs had the capacity to secrete a series of growth factors/cytokines presenting anti-apoptotic, anti-inflammatory, anti-oxidative, and pro-angiogenic effects and modulating host cells. 20 Recent studies have suggested that MSCs may act as an ideal vehicle for subcellular component delivery, offering a new hypothesis on the mechanism underlying cell survival and tissue regeneration under the AKI stressful microenvironment. Subcellular component delivery is a widespread phenomenon observed throughout multiple mammalian cell types. Besides the delivery of small molecules, like proteins, RNAs or ions, over 40 variations of intercellular organelle deliveries have been described. 21,22 Cells under threat from extraneous stress need to maintain their homeostasis and reduce cell injury. Therefore, the acquisition of substances from neighbouring cells seems to be an important adaptation to the variable external environment and a classical example of multicellular cooperation. Emerging evidence indicates that the stress caused by exposure to a cytotoxic microenvironment is a major determinant for subcellular component delivery. Furthermore, the efficiency of the subcellular component delivery doubled when recipient cells were under certain conditions. 23 In some circumstances, cell injury F I G U R E 1 Mesenchymal stem cells' regenerative properties in AKI. It has been hypothesized that MSCs are able to directly differentiate into normal tubular cells or that they hold the capacity to secrete various kinds of cytokines and growth factors, presenting anti-apoptotic, anti-fibrotic, anti-inflammatory and pro-angiogenic effects, promoting renal regeneration. The latter is accepted by most investigators nowadays might even become an entire prerequisite. 24 Compared with the paracrine/endocrine capacity hypothesis, direct subcellular component delivery by MSCs is a faster and more economical strategy for a renal regenerative process, as the biosynthesis of many transferred subcellular components, especially organelles, usually takes a longer time than that that can be afforded by damaged cells in crisis.

| CELLUL AR S TRUC TURE S MED IATING SUBCELLUL AR COMP ONENT DELIVERY IN MSC s
Subcellular component delivery is based on the construction of several kinds of cellular structures between cells. Classically considered intercellular communication processes include ion channels, synaptic vesicles, gap junctions and paracrine receptor-ligand bindings. However, the substances which MSCs deliver in the process of cell regeneration are not only biochemical signals but also defined subcellular components, such as proteins, RNAs and even organelles. In a sense, subcellular component delivery may be considered as a special form of intercellular communication that relies on particular cellular structures. EVs, tunnelling nanotubes (TNTs) and cellular fusion are the three major means for subcellular component delivery ( Figure 2).

| Extracellular vesicles
Extracellular vesicles are a heterogeneous population of biologically active membrane-encompassed vesicles that can be secreted by almost every cell type, including MSCs, to the extracellular medium. 28 According to their origin, size and molecular composition, EVs may be classified in exosomes (EXs), microvesicles (MVs) and apoptotic bodies. Although the components and loading mechanisms are still a matter of debate, it is widely accepted that EVs can act as a shuttle of cargoes, which include lipids, proteins, enzymes, coding and noncoding RNA molecules (eg mRNAs, miRNAs and lncRNAs), and even organelles. 29 After being secreted, EVs can interact with recipient cells, be internalized, and act as donors to release their biologically active molecules inside target cells, making a change on their function. 30 Therefore, EVs can be regarded as envoys for long-distance subcellular component delivery between cells.
Exosomes originated from MSCs (MSC-EXs) (40-100 nm in diameter) are generated from the endosomal network. Due to their small size, it has widely been accepted that they are responsible for transporting small molecular cargoes, such as proteins or genetic components like RNAs. 31,32 MVs derived from MSCs (MSC-MVs) (100-1000 nm in diameter) are formed by outward budding of the plasma membrane in a calcium-and calpain-dependent manner; they are the largest among the different EV types. Consequently, F I G U R E 2 Cellular structures mediating subcellular component delivery in MSCs. MSCs are able to form a series of cellular structures to interact with target cells. EVs and TNTs as well as cellular fusion are the 3 major ones. Various subcellular components, like proteins, mRNAs, miRNAs and mitochondria, can be transferred by these mechanisms besides transporting lipids, proteins, mRNAs and miRNAs, they also undertake the task of organelle delivery. To date, mitochondrial transport is the only well documented and understood organelle transfer mechanism by MSC-MVs. 24 However, in other cell types, it has been demonstrated that MVs can also transfer ribosomes. 33 So, it is plausible to assume that other cytosolic structures may also be transported by MSC-MVs.
In terms of apoptotic bodies, which also constitute a heterogeneous population of EVs, it was found that the perforin-dependent apoptotic process of injected MSCs was essential to initiate MSCinduced immunosuppression in both animal and mankind graft-versus-host disease (GvHD). This fact indicated that engulfed apoptotic bodies also had the ability to deliver subcellular components and explained their therapeutic effects. 34

| Tunnelling nanotubes
In the last 10 years, a burst of attention has been paid on TNTs, a newly discovered form of long-distance cell contact structure.
Morphologically, TNTs are defined as actin-based ultrafine intercellular structures with diameters ranging from 50 to 200 nm and lengths which can span over several cell diameters. 35 Before the determination of their composition, these tubular structures were believed to be extensions of the cell plasma membrane that formed an open-ended conduit between two communicating cells. Biologically, the formation of TNTs seems to facilitate the transmission of cytoplasmic contents, not only of biological molecules, but also of selected organelles. Electrical signals, calcium signalling molecules and proteins are known to be transferred through TNTs. 27,36,37 The motion of multiple organelles through these structures has also been visually confirmed by real-time fluorescence microscopy observation. 38,39 The formation of TNTs has also been widely observed in MSCs.
The vast majority of studies have reported that these structures are mainly established in ex vivo MSC coculture systems. 40 However, a recent study by Li et al confirmed that MSCs could also utilize TNTs to transport mitochondria in an in vivo microenvironment. The transmission of mitochondria through TNTs helped in rescuing lung epithelium cells suffering from cigarette smoke injury. 41 These facts indicated that TNTs are important, commonly found structures involved in MSC-induced intercellular communication.  43 However, based on the fact that few transplanted MSCs could be able to arrive around injured renal cells, the usefulness of this mechanism accounting for MSC-based renal repair treatments still needs to be explored.

| SUBCELLUL AR COMP ONENT DELIVERY PL AYS A CRITI C AL ROLE IN MSC-BA S ED THER APY-MED IATED PROTEC TI ON AG AIN S T AK I
We have demonstrated that there are different cellular structures that mediate subcellular component delivery between MSCs and target cells under different circumstances. Recent studies also found that MSCs were able to utilize this mechanism to alleviate AKI. In the following section, we will discuss about the involvement of subcellular component delivery in MSC-based therapy-mediated protection against AKI.

| Delivery of miRNA
miRNAs are small non-coding RNAs that are able to bind to the 3′ UTR of their target mRNAs, regulating gene expression at a post-transcriptional level. 44 It has been reported that miRNAs are involved in a wide range of biological and pathological processes, including cell proliferation, apoptosis, tumour development and stress response. 44   the reversion of multiple gene alterations in kidneys after injury. 48 Soon, an important question was raised: 'Among the multiple kinds of miRNAs, which one is responsible for the regenerative effect?'. 49 In order to answer this question, Lindoso   Moreover, the expression of the IL-10 protein was detected in HKC8 cells, from which it was originally absent; interestingly, IL-10 mRNA, but not the protein, was initially present in MSC-EVs. These results indicated that IL-10 mRNA was successfully delivered from MSC-EVs to HKC8 cells. Clearly, the acquisition of IL-10 mRNA and its translation into IL-10 protein within HKC8 cells played a role in rescuing cell viability after cisplatin injury. 53 Similarly, Tomasoni et al found that the insulin-like growth factor-1 receptor (IGF-1R) mRNA is selectively shuttled into BM-MDCs EXs. Then, this mRNA can be transferred into cisplatin-damaged PTECs, where it is translated into the IGF-1R protein, stimulating cell proliferation. 54 Based on these ex vivo outcome, some in vivo studies were performed.

| Delivery of mRNA
The hepatocyte growth factor (HGF) is a growth factor that exerts paracrine effects to promote cell regeneration and injury repair in multiple organs and in AKI. 55  Glycerol-AKI is another common animal AKI model where the mechanism of mRNA delivery by MSCs has also been observed.
Human POLR2E mRNA and its translated protein were confirmed to be localized in tubules of mice with AKI treated with MSC-MVs, but not in those from the control group. The accumulation of extraneous components was indeed beneficial for the restoration of the injured renal function. 59 In 2012, a group of researchers confirmed that this phenomenon was also observed in a mice model of cisplatin-AKI, which was also a main type of AKI. 60

| Delivery of proteins
Transferred miRNAs or mRNAs need to be translated into proteins before presenting a biological function. But, is there a direct mechanism for the delivery of functional proteins underlying MSCbased therapy ( Table 1)

| CON CLUS I ON AND FUTURE PER S PEC TIVE S
As discussed in this review, the data mentioned above provide information about a newly discovered mechanism to explain how a acknowledge the funding support from the sources indicated.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.