Adipose-Derived Mesenchymal Stem Cells: A New Tool for the Treatment of Renal Fibrosis

Introduction

Mesenchymal stem cells (MSCs) are found in almost all tissues, except for bone marrow, amniotic fluid, and umbilical cord blood. Adipose tissue has also been identified as an important source of MSCs [1]. MSCs have a strong self-renewal capacity and multidirectional differentiation potential [2]. As a type of seed cell for tissue engineering, MSCs can repair various tissues and organs [3,4]. To date, MSCs have been used in the repair of bone, cartilage, and joint injury [5] and for the treatment of graft-versus-host disease in hematopoietic stem cell transplantation treatment [6], autoimmune diseases [7,8], spinal cord injury, and neurological diseases [9].

In 2001, Zuk et al. successfully isolated MSCs from adipose tissue [10]. Adipose tissue has since received considerable attention as an ideal stem cell source due to its availability, abundance, and self-filling properties [11]. In vitro cultured human adipose-derived mesenchymal stem cells (AMSCs) can grow adherently with fibroblast-like morphology [12]. Under different conditions, AMSCs can be successfully induced to differentiate into mesoderm cells, such as osteocytes, chondrocytes, skeletal muscle cells, liver cells, and cells derived from other germ layers, demonstrating that these cells represent a type of pluripotent stem cells [13,14]. AMSCs cultured in vitro lack specific surface markers [14,15]. Numerous studies confirm that AMSCs do not express the hematopoietic markers CD34 and CD45 and fibroblast markers HLA-DR [16] but highly express CD73, CD90, and CD105 [17,18].

Renal fibrosis is the major pathological change and common pathway of end-stage renal disease [19], and its mechanism of occurrence remains unclear [20]. The occurrence of renal fibrosis is related to the following five aspects. (1) Kidney injury initiates an inflammatory response and infiltration of a large number of various types of inflammatory cells [21], and a variety of inflammatory cells are involved in tubulointerstitial fibrosis, such as lymphocytes, monocytes/macrophages, mast cells, dendritic cells, and endothelial cells. (2) Massive release of fibrosis-related factors, such as cytokines, growth factors, and chemokines [22]. (3) Imbalance of the synthesis and degradation of extracellular matrix (ECM) and excessive accumulation of ECM in the renal interstitium [23]. (4) Mesenchymal transition of innate renal cells and reduction of the number of intrinsic cells [24]. (5) Renal microvascular injury [25]. These five aspects are involved in the process of renal fibrosis, and interventions targeting these aspects can delay or even reverse the progression of renal fibrosis to a certain extent [26].

The regeneration of kidney tissues which is followed by complicated biological regulative mechanism is relative to many factors. AMSCs can produce and release a large number of growth factors, chemokines, and cytokines that modulate intercellular interaction. In fact, these secreted factors can help angiogenesis, stimulate ECM remodeling, reduce inflammation, apoptosis, and oxidative stress, and involved in immune responses. However, these mechanisms do not exist alone but interact with each other. The details have been described below.

AMSCs Homing to the Kidney

Although the homing mechanism of MSCs remains unclear at present, a large number of reports demonstrate that the transplanted MSCs can be nonspecifically distributed in the organs throughout the body, including the lungs, liver, spleen, and kidney, of which the lung tissue is the main interception site [27]. Villanueva et al. have evidence of the arrival of AMSCs into kidney. The presence of renal AMSCs was evaluated by Oct-4 marker. They found that the expression of Oct-4 staining was marked increased at 35 days after AMSCs therapy in 5/6 NPX (nephrectomy) animals [28]. MSCs homing to the damaged kidney can differentiate into innate cells of the kidney or fuse with innate cells of the kidney. In post-renal injury due to inflammation, ischemia, or hypoxia, autologous or exogenous MSCs under the influence of many factors cross the vascular endothelial tissue and can subsequently differentiate into innate cells of the kidney or fuse with innate cells [29] by homing to the damaged kidney tissue with a preferential efficiency of orientation. The injured tissue upregulates the expression of stromal cell-derived factor, platelet-derived growth factor (PDGF), and matrix metalloproteinases (MMPs). The tissue subsequently expresses specific receptors or ligands, triggering the chemotaxis, migration, and adherence of MSCs to the lesion and repair of various kidney damages under appropriate conditions [30]. The therapeutic effects of AMSCs depend on whether stem cells can home to the target organs to exert its function.

We have not found any relevant information about precise localization of autologous or allograft AMSCs after transplantation [31]. Li et al. transplanted human AMSCs into a mouse model of renal ischemia-reperfusion. The transplanted AMSCs could differentiate into tubular epithelial-like cells, confirming that transplanted AMSCs were integrated into the renal tubular structure to replace damaged or necrotic tubular cells. This process may represent one of the mechanisms by which AMSCs directly repair renal tissue damage [32].

Baer et al. [33] demonstrated in vivo that certain signals from renal tubular epithelial cells can induce the transformation of human AMSCs into epithelial cells under certain conditions [34]. After cultured in the conditioned medium derived from tubular epithelial cells (TECs) for 17 days, the expression of three characteristic epithelial markers (cytokeratin 18, ZO-1, and ZO-2) was upregulated during epithelial differentiation of AMSCs [35]. These mutual interactions in cell level may be involved in the repair of kidney damage.

AMSCs Protect Renal Tissues Through Endocrine/Paracrine Mechanisms

AMSCs can directly convert into cells in the damaged kidney to exert renal protection function. More importantly, AMSCs act through the paracrine and endocrine mechanisms to protect renal tissues like other MSCs. AMSCs promote endogenous renal repair by secreting various protective factors. Studies have demonstrated that AMSCs can secrete hepatocyte growth factor (HGF) [36], vascular endothelial growth factor (VEGF) [37], insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), granulocyte–macrophage colony-stimulating factor, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-7, IL-8, and IL-11 in the early cell growth phase [38]. Therefore, the repair effect of AMSCs on the kidneys is mediated not only through the direct differentiation of AMSCs into target cells but also through the complex paracrine mechanism to alleviate the inflammatory reaction of the injured kidneys, playing an important role in the later kidney repair. Stem cell-mediated regulation of these factors can effectively delay the process of renal interstitial fibrosis, alleviate the tissue inflammatory microenvironment, and facilitate renal tubular regeneration and repair.

AMSCs Treat Renal Fibrosis Through Immunomodulation

MSCs are either immune cells or precursors involved in immune regulation, playing a central role in immune activities [39]. Due to the lack of specific surface markers, AMSCs can only be identified by complex immunophenotypes. This therapeutic potential is mediated through a variety of mechanisms, such as regulation of cytokine secretion, activation of regulatory immune cells, and promotion of cell repair via the secretion of antiapoptotic, antifibrotic, and angiogenic factors [40].

A large number of studies have demonstrated that MSCs have low surface expression of human leukocyte antigen (HLA) and co-stimulatory molecules, do not induce proliferation of T lymphocytes when co-cultured with homologous T lymphocytes in vitro, and exhibit low immunogenicity. MSCs do not express the human major histocompatibility antigen MHC-II class molecules and immunocostimulatory molecules, such as CD86 (B7-1), CD40 (Bp50), and CD40L (gp39). However, these cells exhibit low expression of MHC class I molecules [41]. The activation of effector T cells requires the involvement of MHC-II or co-stimulatory molecules as the second signal. MSCs are rendered less immunogenic due to the lack of expression of the second signal molecules. MSC also has certain regulatory effects on immune cells, such as T cells, B cells, natural killer cells, and dendritic cells. This regulation may be mediated by MSCs-secreted soluble regulatory factors, such as IL-10, transforming growth factor-β1 (TGF-β1), prostaglandin E2, HGF, and IL-2 [42].

MSCs derived from different tissues have different immunogenicity given their differential activation states and differences in species, tissue sources, and culture conditions [43]. There are currently no large numbers of specific studies focusing on the low immunogenicity of AMSCs. Puissant et al. compared the immunological properties of AMSCs with bone marrow-derived stem cells (BMSCs) and found that AMSCs can inhibit mitogen-stimulated T cell proliferation and mixed lymphocyte reaction without causing lymphocyte proliferation [44]. Therefore, human AMSCs also exhibit low immunogenicity and immunomodulatory function [45].

AMSCs Interfere with Renal Fibrosis Through Anti-inflammatory Effects

Inflammation is the basic pathological change of chronic kidney disease (CKD) and the initiating factor of renal fibrosis. Inflammation is mainly manifested as immune cell infiltration and secretion of inflammatory mediators. The infiltration of inflammatory cells, inflammatory cytokines, and inflammatory chemokines plays an important role in the pathogenesis of renal interstitial fibrosis. Inflammation is also one of the key causes of renal failure.

AMSCs reduce the epithelial–mesenchymal transition (EMT) through the early regulation of inflammation and hypoxia and the improvement and delay of the fibrosis process [46]. Expanding research has revealed that inflammatory mediators are involved in the process of renal interstitial fibrosis, such as growth factors, interleukin family, TNF-α, and chemokines [47]. AMSCs exhibit chemotactic attraction to several cytokines [48], including PDGF, VEGF, IGF-1, bone morphogenetic protein-4 (BMP-4), and BMP-7 [49]. Among them, BMP-7 is a TGF-β superfamily member and can counteract the profibrotic effect of TGF-β.

The infiltration of macrophages is closely related to the degree of renal injury and fibrosis. Macrophages can be roughly divided into the classically activated M1 macrophages and selective M2 macrophages. Glomerular and interstitial macrophage infiltration consists of macrophages with the classically activated M1 phenotype, which produces a variety of proinflammatory molecules, including IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), IL-12, MMP-12, and other tissue factors [50]. At the stage of proinflammatory chronic fibrosis in kidney disease, macrophages may shift from the M1 to M2 phenotypes. In the chronic phase of renal fibrosis, macrophage loss is significantly reduced.

Eirin et al. demonstrated that AMSCs directly induce the phenotypic conversion of M1/M2 macrophages [51]. After co-culture with AMSCs, macrophages exhibit reduced expressions of M1-related markers, such as iNOS and TNF-α, and increased expressions of M2 phenotype-associated markers, such as IL-10 and arginase-1 (Arg-1) [52]. AMSCs treatment can improve early renal function changes and reduce renal fibrosis progression. In the late stage of injury, EMT is reduced mainly due to the early regulation of inflammatory responses and the reduction of hypoxia.

Extracellular Vesicles of AMSCs Play a Role in the Regulation of Fibrosis

Extracellular vesicles (EVs) are distinguished according to different diameters and mainly consist of microvesicles (MVs), exosomes (Exs), and apoptotic bodies. EVs are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and milk); carry a variety of cell-derived proteins, lipids, DNA, mRNA, and miRNAs; and are involved in processes, such as intercellular signal transduction, cell migration, angiogenesis, and immune regulation.

The surface antigens of AMSCs collected and cultured in laboratories mainly include CD44, CD90, and CD105. The MVs mainly expressed β1-integrins, CD73, and CD40, whereas Exs mainly express CD9 and CD81. EVs of AMSCs selectively accumulate certain mRNAs, such as the angiogenesis-related genes HGF, HES1, and TCF4, adipogenic genes of CEBPA and KLF7, and express Golgi apparatus genes (ARRB1, GOLGA4) and genes involved in TGF-β signaling [53].

Currently, there are few reports on MVs of AMSCs. MVs of MSCs exhibit anti-ischemic acute kidney injury effects [54], reverse the progression of CKD, increase tubular cell proliferation, reduce renal tubular cell apoptosis, and significantly reduce damage to renal function [55]. Regarding angiogenesis stimulation, the probable mechanism may be the delivery of microRNA-31 via MVs from AMSCs to vascular endothelial cells [56]. Kang et al. even evidenced that factor-inhibiting HIF-1 was the target of microRNA-31 in human umbilical vein endothelial cells.

EVs are functional biological products, not randomly produced residues of biofilm structures. EVs may be able to overcome the adverse effects of traditional cell therapy [57]. MVs may be effective in avoiding the cell mutation or carcinogenesis due to conventional cell therapy methods, particularly those that occur after long-term treatment with MSC. Whether the EVs of AMSCs can treat the kidney diseases and improve renal function through some unknown mechanisms still needs more researches.

Comparison of AMSCs and Stem Cells from Other Sources

At present, the most commonly used stem cells are BMSCs. However, due to the low abundance of bone marrow and the low proportion of BMSCs, it is difficult to purify BMSCs efficiently and rapidly [58]. AMSCs are attracting increasing attention given their advantages [59], such as convenient source, minimal injury, good cell viability, rapid proliferation, and easy purification. Functionally, AMSCs exhibit similar biological activity and stronger immunomodulatory function. Thus, AMSCs are more suitable for basic research and clinical applications of cell therapy [60].

Endothelial progenitor cells have also been previously investigated in the improvement of renal interstitial fibrosis. AMSCs also have significant advantages compared with endothelial progenitor cells. Although endothelial cells can reduce oxidative stress by significantly enhancing the expression of growth factors [61], the acquisition process requires a large amount of peripheral blood. In contrast, AMSCs exhibit similar angiogenic properties [62] and can more pronouncedly reduce the inflammatory response, endoplasmic reticulum stress, and apoptosis [63].

Clinical Application and Limitations of AMSCs

The kidney develops from mesoderm. Therefore, mesoderm-derived AMSCs are potentially promising stem cells in the treatment of CKDs [64]. In the past few years, MSC has been successfully used in CKD-related experimental models with certain efficacy, including diabetes [65], hypertension, and chronic renal transplant renal disease [66]. AMSCs have numerous advantages, such as widely available sources, easy access, minimal patient suffering, adequate cell count, and lack of ethical concerns. What's more, AMSCs have been proved not to be affected by renal disease [67]. The biological characteristics of AMSCs in patients with kidney disease are similar to those of normal individuals, and the inflammatory state of uremic patients does not compromise the immune function of AMSCs. This suggests that autologous AMSCs are suitable for the treatment of renal patients. Also, AMSC therapy has been proved in some animal models of kidney diseases that it improves renal function and prolongs the progression of renal fibrosis [68]. Currently, there are many clinical trials about stem cells therapy for kidney disease. Three of these items are related to AMSCs in the treatment of kidney disease. In Mayo Clinic, autologous AMSCs have been effectively used for clinical treatment of renal vascular occlusive disease [69]. There is no uniform standard for injection dose. According to the weight of patients, it is generally 1–3 × 10⁵/kg. The AMSCs are injected into the human body through vein or renal artery. Antioxidation, antiapoptosis, and anti-inflammatory pretreatment are needed before injection.

On the basis of a large number of previous researches, we can conclude that AMSCs can serve as an ideal tool for autologous stem cell treatment. However, long-term in vitro passage of stem cells can lead to phenotypic changes; the lack of specific markers and inefficient extraction of AMSCs may limit its application [70], and its long-term clinical safety and efficacy remain uncharacterized [71]. In early in vitro culture phases, stem cells maintain pluripotency. In contrast, stem cells cultured to the late stage exhibit some signs of aging, and cancerous cells may develop [72]. The immune regulatory mechanisms of stem cell therapy and anti-inflammatory and antioxidative stress mechanisms are not completely understood [73].

Based on these characteristics, the mechanism by which AMSCs can treat renal fibrosis is worthy of further investigation, and cell therapy will become a very valuable research hot spot.