Recent developments in regenerative medicine have provided attractive solutions for the treatment of renal diseases. Several technologies developed in regenerative medicine have been applied to treat renal failure [1-3]. Among the technologies, cell-based therapy has been primarily considered as a therapeutic intervention to provide renal functions [1-3]. A commonly used approach in cell-based therapy is cell transplantation, where therapeutic cells are exogenously administered to achieve the repair. The cell transplantation approach using various cell types have resulted in reno-therapeutic outcomes in many pre-clinical [1-3] and clinical studies [4]. For example, primary renal cells isolated from kidney tissues were introduced to improve renal functions from kidney diseases in a pre-clinical [5] and clinical study [4]. Similarly, various stem cells, including adult, fetal, embryonic, and induced pluripotent stem cells also have been extensively studied as therapeutic cell sources and demonstrated the beneficial therapeutic effects in renal failure [6]. While the cell transplantation approach has demonstrated promising outcomes, in terms of improvement of renal function, several safety issues still remain to be addressed prior to applying clinically. These include immune rejection, pulmonary embolism, and teratoma formation by uncontrolled proliferation of pluripotent stem cells [7,8]. While cellular therapy has become a potential therapeutic modality for selected populations, its mode of action remains controversial. Many investigators believe that the effects of cell therapy may be primarily due to trophic factors secreted by the administered cells (secretomes). Consequently, research using cell-derived secretomes has been actively pursued in various areas, including the kidneys.

Secretome is generally referred to as the trophic factors present in conditioned medium of cell culture (CM) [9], and is comprised of soluble and insoluble molecules enriched with bioactivity that is suitable for tissue regeneration. Several studies reported that exogenous delivery of secretomes resulted in therapeutic efficacy on the improvement of renal functions in pre-clinical and clinical studies [10-13]. The therapeutic outcome by the secretome treatment is largely attributed to various secretome-derived bioactive compounds such as cytokines, chemokines and growth factors, lipids, free nucleic acids, and extracellular vesicles (EV) [14-16]. The important roles of the secretome compounds localized within the tissue injury site are believed to be angiogenesis, modulation of inflammatory, and inhibition of apoptosis of renal cells [11] (Fig. 1). In this article, we will review these secretome components and applications using various types of cell-derived secretomes for treatment of renal diseases (Table 1). Engineering platforms for efficient delivery of the secretomes will also be discussed (Table 1).

While cell secretome has shown to improve renal function in multiple studies, the delivery method has been a limitation to achieve maximum therapeutic effects of the secretome. For instance, Xing et al. reported that MSCsecretome was not effective in improving renal function in an IRI kidney injury model in mice, which may be due to inefficient delivery of the secretome in a low concentration state [63]. Similarly, Abedi et al. observed partial tissue regeneration when MSC-derived secretome was intraperitoneally injected into animals with kidney failure [27]. In either study, the injected trophic factors in the secretome may have lost their biological activity within a short time, likely due to a high diffusion rate and short half-life following injection [64-66]. To address this issue, the controlled release of secretome factors into the injured site is necessary to achieve improved therapeutic effects.

Drug delivery system has been developed as a promising option to facilitate a controlled and sustained release of bioactive factors to exert maximized outcomes in tissue regeneration [67-70]. Delivery vehicles, including natural and synthetic materials, have been designed to physically encapsulate target factors or immobilize the factors on the vehicles, thereby protecting them from the effect of enzymatic environment and prolonging therapeutic effects in vivo [67-70]. As a delivery vehicle, hydrogel systems have been widely used, and a combination of hydrogel with the secretome has been proposed as a possible platform due to its versatility required for biological use [71]. In several studies, secretome encapsulated in the hydrogel was used by directly adding hydrogel-initiating materials into the cell culture. In a study, Bakota et al. developed an injectable multidomain peptide (MDP) nanofiber hydrogel system for delivering secretome [72]. The MDF hydrogel is designed to soak secretome factors when placed into the ESC cell culture. The self-assembled MDP hydrogel loaded with stem cellderived secretome demonstrated stability over time, sheer thinning tolerance, allowing for targeting release condition that can be easily delivered through needle injection. In a release kinetic study in mice, the administered hydrogels with secretome through an intraperitoneal injection remained localized for 24 hours, indicating the effectiveness of secretome delivery via MDP hydrogel system. In a further study, Wang et al. examined whether the MDP nanofiber hydrogel system was able to exert reno-protective effects using lipopolysaccharide (LPS) induced both an in vitro and an in vivo model [73]. LPS exposure resulted in a significant increase in activation of caspase-3, which mediates apoptosis [74], and RhoA/Rho kinase, which involves in cell permeability and apoptosis [75], but the presence of preconditioned nanofibers reduced cell apoptosis in an in vitro by preventing these activations. The introduction of preconditioned nanofibers, which were developed by combining stem cell with the nanofibers, improved LPS-induced kidney in both an in vitro and an in vivo study. In an in vivo study using LPS-induced kidney injury, a rise in serum blood urea nitrogen (BUN) and creatinine levels in the LPS injected mice was significantly reduced when treated with nanofibers hydrogel with secretome. They suggested that the nanofiber hydrogel system could be used as a secretome delivery platform for the treatment of renal failure. More recently, Waters et al. developed a nanocomposite hydrogel system to facilitate secretome-based a dual-action therapy [76]. They used 2D synthetic nanoclay material LAPONITE® nanosilicates to form a complex with growth factors in the secretome [76]. By UV crosslinking of mixtures of nanoclay-secretome and gelatin methacrylate (GelMA), they were able to load the crosslinked complex of nanoclay-growth factors within a nanocomposite hydrogel that potentially deliver secretome released from human MSC. The nanoclay hydrogel showed a controlled release of growth factors such as VEGF and FGF2, by slowing secretome release overtime in an in vitro study. Comparing with nanoclay hydrogel without secretome, the angiogenesis potential of the hydrogel with secretome was confirmed by a significant increase in endothelial cell proliferation and migration observed in a microchannel migration assay and the inhibition of apoptosis was observed in a TdT-mediated dUTP nick end labeling (TUNEL) assay. They suggest that the nanoclay hydrogel system containing pro-angiogenesis and tissue-protective ability has the potential to provide a valuable therapeutic platform for tissue regeneration.

Recently, our group developed a clinically relevant hydrogel-based delivery system to efficiently deliver human placental stem cells (hPSCs)-derived secretome to treat kidney ischemia in rats [77]. Unlike the previous three studies where hydrogel formation with cell secretome occurs on the cell culture in situ through a one-step process [72,73,76], our approach consists of two steps; 1) collection of cell secretome from cell culture; 2) incorporation of the collected secretome into a hydrogel system. Thus, our strategy enables for an “off-the-shelf” therapy and efficient quality control of the secretome by utilizing cryopreserved secretome. As the clinically relevant hydrogel source, plateletrice plasma (PRP) hydrogel was used since PRP is biocompatible and safe for clinical applications [78,79]. Furthermore, autologous PRP obtained from the patient is therapeutically beneficial to minimize immune response as compared with that of an allogeneic or xenogeneic source. In our study, we demonstrated that hPSC-derived secretome contained the pro-proliferative and anti-apoptotic ability for endothelial and primary human tubular cells. A release kinetics study showed that incorporation of secretome within the PRP gel system facilitated controlled release of secretome factors in an in vitro and in vivo study. When the PRP encapsulated secretome was tested in an IRI injury model in rats, the secretome+PRP treated group showed significantly lower serum creatinine (Cr) and neutrophil gelatinase-associated lipocalin (NGAL) levels than that of other groups, demonstrating improvement in renal function compared with other controls. We confirmed that the therapeutic outcomes by the delivery of secretome through PRP gel are due to minimized renal tissue damages through anti-apoptotic and pro-angiogenic capability. Our results suggest that secretome encapsulation with PRP gel could be an effective approach for the controlled delivery of secretomes for recovery of damaged kidneys.

While the cell-derived secretome approach demonstrates promising reno-therapeutic outcomes in pre-clinical and clinical studies [80], a number of challenges need to be addressed before using this modality for clinical translation. A major issue in the use of secretome is attributed to the unidentified characteristics of the cell-secreted factors [7]. Further studies are needed to characterize better and define the secreting factors, which allows for improved control and regulation for clinical translation. Furthermore, clinical efficacy and indication need to be assessed in well-designed and controlled clinical trials [81].

In the manufacturing process for commercialization, production, and concentration of secreted molecules in quantities sufficient for clinical administration are also challenging, in terms of efficient therapeutic effects. A recent study involving the EV production on a large scale [36] would enable efficient delivery of sufficient quantities of EV containing miRNAs could be adapted for clinical translation. Also, it is important to note that differential expression of specific molecules in the secretomes might result in different level of beneficial outcomes. For example, Pires et al. demonstrated different profiles of secretomes from BM-MSC, adipose derived cells (ASC), human umbilical cord perivascular cells (HUCPVCs) using a proteomic analysis [82]. Interestingly, secretion profiles of the specific secretomes from BM-MSC allowed more anti-oxidative stress than that of ASC and HUCPVCs. Interestingly, BMMSC and HUCPVCs showed better anti-apoptotic properties than ASCs, while HUCPVCs is not appropriate as anti-scarring agent. Therefore, appropriate selection of the secretome is needed to maximize therapeutic outcomes for efficient renal treatment.

In addition, the engineering strategy of secretome components has a significant potential to develop a treatment for kidney damage efficiently. For example, engineering of exosome as a therapeutic cue may be a viable approach to improve kidney function and promote regeneration, where exosome could be modified to load therapeutic drugs passively or the exosome surface can be anchored with the targeting moieties to efficiently home to the repair site [83]. Such engineering strategies would be beneficial to enhance therapeutic outcomes compared with the naïve type.