Progress in the application of mesenchymal stem cells to attenuate apoptosis in diabetic kidney disease

Abstract

Diabetic kidney disease (DKD) has a high incidence and mortality rate and lacks effective preventive and therapeutic methods. Apoptosis is one of the main reasons for the occurrence and development of DKD. Mesenchymal stem cells (MSCs) have shown great promise in tissue regeneration for DKD treatment and have protective effects against DKD, including decreased blood glucose and urinary protein levels and improved renal function. MSCs can directly differentiate into kidney cells or act via paracrine mechanisms to reduce apoptosis in DKD by modulating signaling pathways. MSC-derived extracellular vesicles (MSC-EVs) mitigate apoptosis and DKD-related symptoms by transferring miRNAs to target cells or organs. However, studies on the regulatory mechanisms of MSCs and MSC-EVs in apoptosis in DKD are insufficient. This review comprehensively examines the mechanisms of apoptosis in DKD and research progress regarding the roles of MSCs and MSC-EVs in the disease process.

Key Words: Apoptosis; Diabetic kidney disease; Mesenchymal stem cells; miRNA; Extracellular vesicles

Core Tip: Diabetic kidney disease (DKD) represents a prevalent and serious complication of diabetes. Mesenchymal stem cells (MSCs) and their extracellular vesicles (MSC-EVs) show promise for treating DKD by regulating apoptosis; however, challenges such as a lack of standardization, hinder their clinical application. This review examines the research progress on the roles of MSCs and MSC-EVs in this disease.

INTRODUCTION

Diabetic kidney disease (DKD) results from long-term chronic disorders of glucose and lipid metabolism and is a prevalent chronic microvascular complication of diabetes[1].

In 2021, 10.5% of the global population (536.6 million individuals) had diabetes. By 2045, this figure is projected to increase to 12.2% (780 million patients), with type 2 diabetes accounting for approximately 90% of the total incidence[2]. Approximately 20%-40% of patients with diabetes develop DKD[3], which is the primary cause of end-stage renal disease[4,5].

DKD can be caused by multiple factors, such as impaired insulin signaling and metabolic disorders, which can oxidize mitochondria and produce substantial quantities of reactive oxygen species (ROS)[6]. When ROS accumulate in renal cells, they trigger apoptosis of glomerular and tubular cells, leading to matrix reorganization and tissue fibrosis, thus facilitating the development of DKD[7]. Currently, effective methods for preventing and delaying DKD progression are lacking[8]. In recent years, many researchers have suggested that apoptosis or imbalanced death of renal parenchymal cells is one of the main causes of DKD development[9].

Mesenchymal stem cells (MSCs), a type of adult stem cell endowed with a remarkable self-renewal capacity and multi-directional differentiation potential, possess biological characteristics such as immune regulation, low immunogenicity, inflammatory chemotaxis, and tissue repair[10]. They are promising for tissue regeneration in the treatment of DKD and have certain protective effects, including reduced blood glucose and urinary protein levels and improved renal function[11]. Extracellular vesicles (EVs) derived from stem cells alleviate apoptosis and DKD symptoms by transferring miRNAs to target cells or organs, thus making them safer and more efficacious than cell transplantation[12]. However, research on the regulatory mechanisms of MSCs and their EVs in apoptosis in DKD is insufficient. Therefore, this study summarizes the mechanisms of apoptosis in DKD and research progress regarding the functions of MSCs and their EVs in the aforementioned process.

To systematically evaluate the mechanisms by which MSCs regulate apoptosis in DKD, we searched the literature published in PubMed, Web of Science, Embase, and the Cochrane Library from January 2000 to January 2025. We constructed Boolean operator-based search formulas using keywords and synonyms: (“MSCs” or “mesenchymal stem cells”) and (“diabetic kidney disease” or “DKD” or “diabetic nephropathy”) and (“apoptosis” or “programmed cell death”) and (“therapeutic mechanism” or “exosome” or “miRNA”). Search terms were expanded to include MeSH terms, such as “mesenchymal stem cell” (MeSH), in PubMed and free-text variants, including “caspase-3”, “Bax/Bcl2 ratio”, “TUNEL assay”, and other biomarkers related to apoptosis. The inclusion criteria were as follows: Original research explicitly investigating the regulation of apoptotic mechanisms in MSCs or extracellular vesicle-mediated DKD cells, studies on apoptosis-related signaling pathways or biomarkers, and peer-reviewed articles with full texts in English.

DKD AND APOPTOSIS

Apoptosis, also known as programmed cell death, is pivotal in DKD initiation and progression. This is a highly regulated process. Apoptosis primarily occurs through endogenous, exogenous, or endoplasmic reticulum (ER) stress-induced pathways, mediated by cysteine proteases (caspases)[13] (Figure 1). The overproduction of advanced glycation end products and ROS caused by hyperglycemia can trigger these three pathways, which interact with each other through the mitochondria, thus reflecting an important mechanism that leads to DKD[14,15]. Common signaling pathways that affect apoptosis in DKD include phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) and Wnt/β-catenin pathways (Figure 2).

Figure 1 Pathways of apoptosis. A: Mitochondrial pathway: When the inside of the cell is stimulated, pro-apoptotic proteins such as Bax and Bak are activated and inserted into the mitochondrial membrane, increasing the permeability of the mitochondrial membrane and releasing cytochrome C into the cytoplasm. Cytochrome C combines with Apaf-1 to form an apoptosome, which recruits and activates Caspase-9, and then activates downstream effector caspases, leading to apoptosis; B: Death receptor pathway: Apoptotic signaling molecules outside the cell, such as tumor necrosis factor-α and FasL, bind to the death receptors on the cell surface to form a death-inducing signaling complex. After Caspase-8 is activated, it directly activates downstream effector caspases to trigger apoptosis. It can also activate the endogenous mitochondrial pathway by cleaving the Bid protein to amplify the apoptotic signal; C: Endoplasmic reticulum pathway: When the endoplasmic reticulum undergoes stress due to the accumulation of misfolded proteins, etc., it initiates the unfolded protein response. Through pathways such as the PERK-eIF2α-ATF4 pathway and the IRE1α pathway, it induces the expression of pro-apoptotic genes. At the same time, the endoplasmic reticulum releases calcium ions into the cytoplasm and activates relevant enzymes, such as Caspase-12, etc., ultimately inducing apoptosis. TNF: Tumor necrosis factor. It was drawn using Figdraw software.

Figure 2 Common signaling pathways affecting apoptosis: Phosphatidylinositol 3-kinases/protein kinase B/mammalian target of rapamycin signaling pathway and wnt/β-catenin signaling pathway. PI3K: Phosphatidylinositol 3-kinases; AKT: Protein kinase B; mToR: Mammalian target of rapamycin; GSK-3β: Glycogen synthase kinase-3β; Cyto C: Cytochrome C; APC: Adenomatous polyposis coli gene protein; CK1: Casein kinase 1; Dvl: Dishevelled; TCF: The nuclear T cell factor; LEF: Lymphoid enhancer binding factor. It was drawn using Figdraw software.

The activation of the PI3K/AKT/mTOR signaling pathway suppresses apoptosis and stimulates cell differentiation and survival, and this pathway plays crucial roles in the prevention and treatment of diabetes[16]. PI3Ks are a family of intracellular lipid-protein kinases that regulate cell proliferation, differentiation, apoptosis, and glucose transport[17-19]. PI3Ks can be activated by insulin receptors, growth factors, and external stimuli to generate the second messenger PIP3, which recognizes and binds to Akt, thus changing its conformation. Akt phosphorylation in turn activates the mTOR[20]. Glycogen synthase kinase-3β (GSK-3β) is a downstream protein of Akt that is widely involved in the regulation of mitochondrial function[21]. In DKD, Akt phosphorylation is impeded, leaving GSK-3β in an activated state[22]. Once activated, GSK-3β modulates the Bax/Bcl2 ratio, which subsequently affects mitochondrial membrane permeability and promotes the release of cytochrome C (Cytc) from the mitochondria, which ultimately plays a role in regulating apoptosis[23,24]. Cytoplasmic Cytc initiates apoptosis by activating the apoptosis promoter caspase 9, which cleaves and hydrolyzes downstream members of the caspase family, such as caspase 3[25].

The Wnt/β-catenin axis is a well-studied signaling pathway that affects apoptosis in DKD[26]. When β-catenin protein levels are low, the Wnt pathway is shut down and β-catenin gets phosphorylated by the β-catenin degradation complex[27]. The complex is composed of adenomatous polyposis coli gene protein, axin, GSK-3β, and casein kinase 1[28]. When β-catenin levels increase, the Wnt pathway is activated. The Wnt protein then binds to Fzd and LRP5/6 and recruits the cellular cytoplasmic protein dishevelled, which inhibits GSK-3β activity and prevents the formation of degradation complexes[29]. β-catenin then undergoes dephosphorylation, accumulates in the cytoplasm, and then translocates into the nucleus, where it forms a complex with the nuclear T cell factor/Lymphoid enhancer binding factor and activates a series of downstream genes, such as cyclin D1 and c-myc. These genes regulate the expression of other genes and influence cellular activities, such as proliferation and apoptosis[29]. Previous studies have indicated that the level of β-catenin in the cytoplasm is extremely low in normal kidneys. However, renal injury can trigger the activation of β-catenin in tubular epithelial and mesangial cells[30]. The activation of β-catenin can alleviate apoptosis in DKD, and Nrf2 may be a crucial element in its regulatory route[31].

MECHANISM OF THE AMELIORATION OF RENAL INJURY BY MSCS

MSCs, which are multipotent stem cells that originate from the mesoderm during the early developmental stage, can be derived from various sources, including bone marrow (BM), placenta, adipose tissue, umbilical cord, and urine. MSCs possess remarkable self-renewal abilities, potential for multidirectional differentiation, and low immunogenicity[32]. Additionally, they can generate a diverse range of cytokines and growth factors that exhibit functions such as hematopoiesis support, immunomodulation, and anti-inflammation[33]. MSCs are promising candidates for tissue regeneration in treating DKD[34]. Numerous studies have shown the therapeutic effects of MSCs on DKD, including the attenuation of oxidative damage[35] and inflammatory responses[36,37], inhibition of apoptosis[38] and fibrosis[39,40], modulation of autophagy[41,42], and maintenance of pedicle cell morphology[43]. Currently, MSCs are believed to act on DKD in various ways, including direct differentiation into damaged tissue cells and induction of paracrine effects.

MSCs differentiate directly into injured tissue cells

Prior studies have shown that MSCs have a selective homing effect and can differentiate into various kidney cell types[44], including mesangial cells[45], endothelial cells[46], and epithelial cells[47], thus improving injured kidney tissue and delaying the progression of renal fibrosis[48]. Yokoo et al[49] reported that human BM-derived MSCs (BM-MSCs) could to differentiate into mature kidney structures after being implanted into rat embryos and transplanted into rats. Lee et al[50] found that while undetectable in the lungs, liver, spleen, and other organs, BM-MSCs could colonize the pancreas and kidneys of experimental mice; 11% of the cells migrated to the kidneys. This attenuated the proliferation of glomerular thylakoid membranes and decreased macrophage infiltration.

Furthermore, Ito et al[45] discovered that BM-MSCs can differentiate into glomerular mesangial cells in mice. Additionally, Wong et al[51] co-cultured MSCs with oxidatively damaged glomerular mesangial cells in vitro, analyzed their contractility using flow cytometry, and found that MSCs co-cultured with damaged glomerular mesangial cells had a morphology similar to that of glomerular mesangial cells and contracted upon exposure to Ang II. The CD54 (-) CD62E (+) expression on MSCs in this experiment was directly opposite to that of CD54 (+) CD62E (-) on pure MSCs; therefore, it was concluded that MSCs could differentiate into glomerular mesangial cells.

MSCs improve renal injury through paracrine mechanisms

Biologically active molecules secreted by MSCs: One of the most crucial mechanisms by which MSCs improve renal injury is through paracrine function[52]. Chen et al[53] suggested that BM-MSCs mitigate renal injury by secreting related factors that inhibit inflammatory responses and oxidative damage and enhance vascular regeneration through a paracrine mechanism. Park et al[54] discovered that upregulation of the extracellular matrix and epithelial-mesenchymal transition (EMT) could be influenced by providing conditioned medium to rat kidney cells (NRK-52E) without contact with human umbilical cord blood stem cells (hUCB-MSCs), suggesting that hUCB-MSCs could prevent and control DKD through paracrine effects. Numerous studies have shown that MSCs can secrete various biologically active molecules that can be primarily grouped into three categories: Growth factors, cytokines, and chemokines[55,56]. These molecules, including epidermal growth factor (EGF), hepatocyte growth factor, bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF), interact and play important roles in treating DKD[57-60].

MSCs secrete EVs: Paracrine secretion by MSCs, of which EVs are the main carriers, has been well documented[61]. MSCs secrete EVs (MSC-EVs) protect the kidneys against damage via multiple mechanisms, including autonomous targeting and anti-apoptotic, anti-inflammatory, antioxidant, and antifibrotic mechanisms[62,63]. Exosomes, a type of EVs with diameters ranging from 30 to 150 nm that are generated within intracellular vesicles, incorporate 350-400 proteins derived from the cytoplasmic membrane[64], and represent a unique mechanism for intercellular communication[65,66]. EVs are found in all types of biological fluids (including blood, cerebrospinal fluid, urine, and tear fluid)[66]. Under physiological conditions, EVs are regulated by donor cells, exchange or transmit information to recipient cells in healthy tissues[67] and influence disease progression[68]. EVs can uniquely transmit and exchange intracellular chemical information, interact with recipient cells, and have been applied in the treatment of various diseases[69,70].

The contents of EVs include signaling molecules and proteins, such as mRNA, miRNA, and other small molecules[71,72], which can be delivered to specific cells, regulate intercellular communication, and induce genetic and epigenetic changes in the target cells[73]. miRNAs are important mediators of EVs that perform distinct biological functions[72]. MSC-EVs contain miRNAs that are closely related to cell survival, differentiation, and immune regulation, including miR-223, miR-564, and miR-451[60]. Notably, these miRNAs exert their biological effects by altering the activity of target cells through various mechanisms[74].

MSC-EVs share similar properties with the MSCs from which they are derived and can perform unique biological functions, such as inducing anti-inflammatory, regenerative, and immunomodulatory activities to promote angiogenesis[75]. MSC-EVs express tetrameric molecules (including CD9, CD63, CD81, and CD107) and MSC markers (CD29, CD73, CD44, and CD105), which are intimately linked to the function of MSC-EVs and are capable of interacting with numerous cell types at neighboring/distant sites and eliciting appropriate cellular responses[76]. MSC-EVs are vital for facilitating the capacity of MSCs to function as matrix-supporting cells, maintain tissue homeostasis, and react to external stimuli[77]. Compared to MSCs, MSC-EVs have the following advantages: Smaller size, stronger targeting, lower cytotoxicity, and potential as a new type of nanomedicine carrier for the treatment of various diseases[78]. MSC-EVs cannot self-replicate; hence, the risk of endogenous tumor formation is remarkably reduced. They also exhibit lower immunogenicity and produce fewer embolisms after intravenous injection, making them safer than MSCs[15]. Furthermore, MSC-EVs possess longer circulating half-lives, high permeability, good biocompatibility, and easier quantification and maintenance of biological activity during storage and transportation. MSC-EVs also have important clinical applications in the treatment of various human diseases (Figure 3).

Figure 3 Sources of mesenchymal stem cells and mechanism of the amelioration of renal injury. MSCs: Mesenchymal stem cells. It was drawn using Figdraw software.

THE ROLE OF MSCS AND MSC-EVS ON APOPTOSIS IN DKD

The role of MSCs on apoptosis in DKD

In recent years, several studies have confirmed that MSCs from different sources can protect renal structures and improve renal function by preventing damaged tubular epithelial cells and podocytes from undergoing abnormal apoptosis (Table 1).

Table 1 Role of mesenchymal stem cells on apoptosis in a high glucose cell models and animal models of diabetic kidney disease.

Cell types	Animal models/cell	Dose	Injection method	Frequency/time	Outcomes	Ref.
BM-MSCs	STZ-induced SD rats	1 × 106 cells	Tail vein	Once	↑VEGF, Bcl2; ↓TNF-α, TGF-β, Bax	[59]
BM-MSCs	Podocytes	Transfected with miR-124a	Co-culture	24 hours	↓Apoptosis rate, ↓Bax, caspase-3, ↑Bcl2	[79]
BM-MSCs	STZ-induced rats	3 × 106 cells with miR-124a 4 ng/mm3	Tail vein	Once	↓Bax, caspase-3, ↑Bcl2	[79]
BM-MSCs	STZ-induced male C57BL/6 mice	1 × 104 cells	Tail vein	Twice	↓Apoptotic cells, Bax, ROS; ↑ Bcl2	[82]
BM-MSCs	STZ-induced SD rats	2 × 107 cells	Intraperitoneally	One time at 4 weekly intervals	↑ATF3, Bcl2; ↓Fas, FasL, P53, caspase-3, BAX,BAX/BCL -2	[83]
BM-MSCs	High-fat diet and STZ administration	2 × 106 cells	Tail vein		↑LAMP2; ↓β-cell apoptosis	[84]
AD-MSCs	STZ-induced SD rats	1 × 107 cells	Tail vein	Once	↑Bcl2, klotho, ↓Bax, Wnt1, Wnt3a, Snail, β-catenin	[38]
AD-MSCs	STZ-induced SD rats	5 × 106 cells	Tail vein	Five times at 4 weekly intervals	↑WT1, ↓disruption of the tubules and interstitium	[85]
AD-MSCs	MPC5	1 × 105 cells/cm2	Cultured with hAd-MSC-conditioned medium	24, 48, 72 hours	↓Cleaved caspase-3; Maintain the normal arrangement of synaptopodin and nephrin in podocytes	[43]
hUC-MSCs	STZ-induced SD rats	2 × 106 cells	Tail vein	Once a week for 2 weeks	↓Apoptosis rate, ↓Bax, hioredoxin-interacting protein, ↑Bcl2	[89]
hUC-MSCs	STZ-induced SD rats	2 × 106 cells	Tail vein	three times every 10 days	↓TUNEL + cells, ↓caspase-3, ↑Bcl2/ Bax	[90]

TNF: Tumor necrosis factor; BM-MSCs: Bone marrow-derived mesenchymal stem cells; VEGF: Vascular endothelial growth factor; SD: Sprague Dawley; AD-MSCs: Adipose-derived mesenchymal stem cells; hUC-MSCs: Human umbilical cord mesenchymal stem cells; TGF: Transforming growth factor.

BM-MSCs were the first and most frequently used cells to study apoptosis in DKD. Abdel Aziz et al[59] employed a rat model of STZ-induced diabetes and showed that BM-MSCs improved kidney function, regenerated kidney tissues in rats, reduced the expression of the apoptotic gene Bax, and boosted the expression of the anti-apoptotic gene Bcl2. Furthermore, Sun et al[79] injected mouse BM-MSCs via the tail vein into diabetic rats and found that BM-MSCs combined with miR-124 significantly upregulated the expression of nephrin, podocin, CD2AP, and Bcl2 and inhibited the expression of caspase 3 and Bax. These findings suggest that BM-MSCs combined with miR-124 promote podocyte proliferation, inhibit apoptosis, and suppress the Notch signaling pathway in podocytes. VEGF is one of the most significant factors contributing to podocyte integrity[80]. Researchers have previously applied MSC treatment to an animal model of puromycin-induced podocyte injury by intraperitoneal injection for 60 days and found that BM-MSCs administration restored VEGF expression in the kidneys of mice[81]. Konari et al[82] found that BM-MSCs could ameliorate apoptosis in DKD via mitochondrial translocation, and BM-MSC-derived isolated mitochondria augmented the expression of Bcl2 and suppressed ROS generation in vitro. After the intraperitoneal injection of BM-MSCs into type 1 diabetic mice, Khamis et al[83] observed that BM-MSCs led to a significant reduction in the expression of the pro-apoptotic markers Fas, FasL, and Bax and remarkably increased the expression of the anti-apoptotic marker Bcl2. Additionally, Zhao et al[84] reported a significant increase in the co-localization of mitochondria and LC3-positive autophagosomes after treating INS-1 cells with BM-MSCs using immunofluorescence analysis. This observation suggests that BM-MSCs can improve mitochondrial function by removing damaged mitochondria from chronically hyperglycemic INS-1 cells.

Adipose-derived MSCs (AD-MSCs) are increasingly used in DKD research. In STZ-induced diabetic rat kidneys, AD-MSCs decreased the elevated expression of Bax, Wnt1, Wnt3a, and β-catenin, enhanced the expression of Bcl2, and ameliorated renal cell apoptosis by modulating the Wnt/β-catenin signaling pathway[38]. AD-MSCs were subsequently co-cultured with high glucose-stimulated rat renal tubular epithelial cells (NRK-52E), and it was found that AD-MSCs mitigated apoptosis through the inhibition of the Wnt/β-catenin pathway[38]. Zhang et al[85] reported that multiple intravenous injections of AD-MSCs markedly reduced glomerular hypertrophy and decreased urinary protein levels in diabetic rats. Further studies have confirmed that AD-MSCs attenuate HG-induced damage to podocytes and reduce podocyte apoptosis.

Owing to their terminally differentiated state, podocytes possess a restricted capacity for cell division and have limited regeneration following injury, depletion, and senescence. Furthermore, excessive podocyte loss makes patients more prone to glomerular diseases[86]. MSCs have become potential podocyte-protective agents for cell-based therapies to treat podocyte injury in DKD or other chronic kidney diseases owing to their regenerative and paracrine characteristics[87]. Li et al[43] treated mouse podocytes (MPC5) with human AD-MSCs and found that the high level of EGF in the conditioned medium of MSCs was a key factor in preventing damage and apoptosis of podocytes, reducing the level of cleaved caspase 3 and maintaining the normal arrangement of synaptopodin and nephrin in podocytes. Additionally, when an anti-EGF antibody was added to the conditioned medium of MSCs, it impeded their protective effect on podocytes.

Human umbilical cord MSCs (hUC-MSCs) are regarded as a prospective cell type in regenerative medicine because of their availability, low immunogenicity, absence of ethical disputes, and high proliferative potential[88]. Chen et al[89] assessed the impact of hUC-MSCs on diabetic rats and discovered that hUC-MSCs decreased the apoptotic rate of renal cells. Notably, this outcome was achieved by enhancing Bcl-xL expression and activating apoptosis signal-regulated kinase 1 and p38 MAPK. In our previous study, we treated STZ-induced diabetic Sprague Dawley (SD) rats with hUC-MSC via tail vein injection and found that hUC-MSCs could lower the expression of caspase 3 and Bax/Bcl2 and upregulate the expression of Nrf2 and its downstream factors in diabetic rat kidney tissues. We also found that hUC-MSCs reduced DKD-related oxidative damage and apoptosis by activating Nrf2 in vitro[90].

Several studies have shown that autophagy and ER stress are critical for allowing podocytes to cope with injury. Therefore, podocyte autophagy has emerged as a promising therapeutic target in DKD treatment. Liu et al[91] found that placenta-derived MSCs (P-MSCs) have significantly upregulated expression of the autophagy-related regulators, SIRT1, FOXO 1, LC3 II, and beclin 1, in rat renal tissues. This occurred after 8 consecutive weeks of tail vein injection of P-MSCs into type I diabetic rats, which was attenuated by the autophagy inhibitor 3-MA.

In summary, MSCs from different sources inhibit apoptosis in DKD.

The role of MSC-EVs on apoptosis in DKD

Recent studies have found that MSCs contribute to disease amelioration, mainly through the paracrine pathway of EVs, particularly through MSC-derived exosomes. These exosomes can transfer renoprotective miRNAs to damaged mesangial cells and podocytes, thereby exerting beneficial effects in DKD[70]. The relevant studies associated with these findings are presented in Table 2.

Table 2 Role of extracellular vesicles derived from stem cells on apoptosis in a high glucose cell models and animal models of diabetic kidney disease.

Exosome source	Animal models	miRNA	Dose	Injection method	Frequency/time	Outcomes	Ref.
BM-MSCs	Renal tubular epithelial cells		5.3 × 107	Co-culture	96 hours	↓TGF-β 1, ↓Apotpsis, ↑ZO-1	[92]
BM-MSCs	HUVECs	miR-146a-5p	100 μL	Co-ulture	48 hours	↑IL-10, ↓apoptosis rate, NF-KB	[93]
AD-MSCs	MPC5	miR-486	25 μg/mL	Co-culture	48 hours	Cleaved caspase-3↓	[94]
AD-MSCs	db/db	miR-486		Tail vein	12 weeks	↓Cleaved caspase-3, ↓p-mTOR	[94]
AD-MSCs	MPC 5	miR-15b-15p	25 μg/mL	Co-culture	24 hours	↑Caspase3, Bcl2, ↓cleaved caspase3	[96]
AD-MSCs	SD rats with type 1 diabetes	MiR-125a	50 μg	Tail vein	Twice a week for 3 weeks	↓Bax, ↑Bcl2, ET1↓	[97]
AD-MSCs	MP5 cells	miR-26a-5p	25 mg/mL	Co-culture	48 hours	↓Cleaved caspase-3, Bax↓ Bcl2↑	[98]
AD-MSCs	db/db mice	miR-26a-5p		Tail vein	Once at 13 weeks	↓TUNEL + cells; ↓cleaved caspase-3, Bax; ↑Bcl2	[98]
HUC-MSCs	Podocytes		30 μg/mL	Co-culture	48 hours	↑Podocytes cell viability, ↓apoptosis of podocytes	[99]
HUC-MSCs	HK2 cells	miR-424-5p	50 μg/mL	Co-culture	48 hours	↓Apoptosis rate, ↓TUNEL + cells, ↓Bax,cleaved caspase-3/caspase-3, ↑Bcl2	[100]
HUC-MSCs	db/db	miR-424-5p	10 mg/kg bw	Tail vein	twice a week for 6 weeks	↓Bax, cleaved caspase-3/total caspase-3, ↑Bcl2	[100]
HUC-MSCs	STZ-induced SD rat	Mi-R-146a-5p	2 × 106	Tail vein	Once a week for 2 weeks	↑IL-10, M2 macrophage marker; ↓IL-1β, IL-6 and TNF-αmRNA, TRAF6, M1 macrophage marker	[101]
HUC-MSCs	Type 2 diabetic BKS. db/db mice	MiR-22-3p	Exo 20, μg/mL	Tail Vein	3 times in the first week, twice a week in the next 3 weeks	↑wnt-1; ↓ Caspase-1, ASC	[102]
HUC-MSCs	Podocytes	MiR-22-3p	20 μg/mL	Co-culture	24 hours	↑Podocytes cell viability; ↓apoptosis of podocytes; ↓Caspase-1	[102]
USCs	STZ-induced SD rat	VEGF, TGF-β1, angiogenin, BMP-7	100 μg	Tail vein	Weekly for 12 weeks	↓Apoptosis rate; ↓TUNEL + cells; ↓caspase-3	[103]
USCs	HPDCs	miR-16-5p	0.4 × 105 cells	Co-culture	4-5 days	↑Podocyte viability; ↓apoptosis rate, Bax, Caspase-3, α-SMA, VEGFA	[104]
USCs	STZ induced SD rat	miR-16-5p	100 μg	Tail vein	once a week for 12 weeks	↓VEGFA, MCP-1, TGF-β1, TNF-α	[104]
USCs	STZ-induced SD rat		2 × 106 cells	Tail vein	Six times every other week	↓TUNEL + cells; ↓caspase-3	[105]

BM-MSCs: Bone marrow-derived mesenchymal stem cells; TGF: Transforming growth factor; HUVECs: Human umbilical vein endothelial cells; AD-MSCs: Adipose-derived mesenchymal stem cells; mTOR: Mammalian target of rapamycin; SD: Sprague Dawley; hUC-MSCs: Human umbilical cord mesenchymal stem cells; USCs: Urothelial stem cells; BMP-7: Bone morphogenetic protein-7; TNF: Tumor necrosis factor.

Notably, Nagaishi et al[92] administered BM-MSCs and MSC-conditioned medium (MSC-CM) to diabetic mice and found that both interventions reduced proteinuria. EVs purified from the conditioned medium of BM-MSCs exhibited anti-apoptotic effects and safeguarded the tight junction structure of renal tubular epithelial cells, indicating that MSC therapy holds promise as a means of preventing DKD through paracrine effects. Furthermore, Chen et al[93] found that AS-IV treatment promoted MSC-exo secretion and augmented the expression of miR-146a-5p. When MSCs-EVs with high miR-146a-5p expression were transferred to HG-damaged human umbilical vein endothelial cells (HUVECs), an improvement in cell viability and tube-forming ability was noted. Simultaneously, the apoptosis rate and inflammatory response decreased. After HUVECs were treated with the MSC miR-146a-5p inhibitor, the mRNA and protein levels of tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and NF-κB were markedly increased. These observations indicate that miR-146a-5p restrains TRAF6 and phosphorylated NF-κB expression. Additionally, Ebrahim et al[42] reported that exosomes derived from BM-MSCs can trigger autophagy via the mTOR signaling pathway, thereby ameliorating renal damage in a type 1 DKD rat model.

Jin et al[94] found that AD-MSC-EVs suppressed the Smad1/mTOR signaling pathway in podocytes by elevating the expression of miR-486, thus leading to an increase in autophagy, decrease in podocyte apoptosis, and improvement in DKD symptoms. Furthermore, a recent study showed that AD-MSCs-EVs are capable of delivering miR-215-5p to podocytes and inhibiting high glucose-induced accumulation of ZEB2 by directly targeting the 3’-UTR of ZEB2, thus weakening HG-induced podocyte EMT and eliciting a sequence of pathological alterations that eventually relieve the symptoms of DKD[95]. Furthermore, Zhao et al[96] found that miR-15b-5p suppression counteracted the protective effect of AD-MSCs-EVs on podocytes in a high-glycemic milieu, suggesting that AD-MSCs-EVs protect podocytes in a high-glycemic milieu by enhancing miR-15b-5p expression. Additionally, PDK4 expression increased after inhibiting miR-15b-5p expression, and knockdown of PDK4 resulted in decreased apoptosis and reduced inflammation in podocytes in a high-glucose environment. These findings suggest that miR-15b-5p protects podocytes in a high-glucose environment by downregulating its target gene, PDK4. MiR-15b-5p modulates the expression of VEGF-A via PDK4[96]. Moreover, inhibition of VEGFA can mitigate the injury of MPC5 cells in a high-glucose environment by decreasing inflammation and apoptosis[96]. Hao et al[97] showed that AD-MSC-EVs regulate apoptosis in type 1 DKD cells through miRNA-125a. Duan et al[98] found that AD-MSC-EVs reduce the apoptosis of podocytes induced by high glucose stimulation. After applying them to db/db mice, it was found that AD-MSCs-EVs rich in miR-26a-5p could target TLR4 and inhibit the NF-κB/VEGFA pathway, thus suppressing cell apoptosis and alleviating the damage to podocytes in db/db mice.

Additionally, Wang et al[99] using both in vivo and in vitro studies, discovered that elevated glucose levels could induce apoptosis and simultaneously activate the nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) signaling pathway in podocytes. HUC-MSC-EVs inhibit the NOD2 signaling pathway by inhibiting the phosphorylated forms of its downstream effector molecules, namely phosphorylated receptor-interacting protein 2 and NF-κB p65. Thus, they alleviate the apoptosis and inflammation induced by high glucose. Cui et al[100] found that hUC-MSC-EVs inhibit hyperglycemia-induced apoptosis and EMT in HK2 cells and db/db mice and express miR-424-5p. They transfected HUC-MSCs with an miR-424-5p inhibitor and then extracted exosomes. They also found that miR-424-5p expression was reduced and could not reduce apoptosis, EMT, and the expression of Yes-associated protein 1 (YAP1). Their research presented fresh perspectives on the protective function mediated by hUC-MSC-EVs in DKD, where hUC-MSC-EVs were able to suppress high-glucose-induced apoptosis and EMT by miR-424-5p targeting of YAP1.

Zhang et al[101] injected hUC-MSCs into STZ-treated male SD rats through the tail vein and found that they restored miR-146a-5p expression in rat kidneys. MiR-146a-5p derived from hUC-MSCs inhibits renal inflammation and restores renal function by targeting the TRAF6-STAT1 pathway and promoting M2 macrophage polarization. Additionally, Wang et al[102] showed that hUC-MSC-EVs attenuate podocyte apoptosis, as well as the levels of NLRP3 and proteins in its downstream signaling pathway, in high-glucose-treated samples and diabetic mice. When miR-22-3p was knocked down in MSC-EVs, its anti-apoptotic and anti-inflammatory functions were eliminated both in vitro and in vivo, suggesting that EVs containing miR-22-3p derived from hUC-MSCs could be targeted to suppress the activation of NLRP3 signaling and reduce apoptosis and inflammation of podocytes in DKD.

Jiang et al[103] used an STZ-induced diabetic rat model to demonstrate that urothelial stem cells (USCs) reduced urinary protein content, decreased caspase 3 expression, and attenuated apoptosis of glomerular podocytes. They also showed that USC intervention could inhibit podocyte apoptosis in a cellular model of podocyte injury induced by high-glucose culture medium. The researchers further examined the expression of cytokines in USCs-conditioned medium and USCs-exo and found that the expression of VEGF, transforming growth factor-β, angiopoietin, and BMP-7 was notably higher in USCs-conditioned medium than in the control medium in which USCs were not cultured. USC-exo has been shown to inhibit VEGFA by overexpressing miR-16-5p, promoting podocyte proliferation, and inhibiting podocyte apoptosis, thus protecting podocytes from DKD damage[104]. Dong et al[105] used to STZ-induced diabetic rats and found that USC intervention improved urinary protein and renal function, reduce caspase 3 expression, and attenuated apoptosis in the renal tissues of diabetic rats.

Collectively, MSC-derived EVs from heterogeneous sources exert regulatory effects on apoptotic pathways in diabetes models, with their therapeutic efficacy largely attributable to miRNA-dependent mechanisms.

PERSPECTIVES ON THE FUTURE APPLICATIONS OF MSCS

Although this study highlights MSC-mediated suppression of apoptosis, recent findings suggest that pyroptosis[106] and ferroptosis[107] may contribute to DKD progression. Further work should explore whether MSC-derived exosomes modulate these pathways to achieve broader protective effects against DKD.

Despite the promising preclinical evidence, multiple obstacles impede the clinical use of MSC therapy for DKD[108]. First, up to now, the majority of studies have been carried out on rodent models, with limited data from large animal studies, such as primates or pigs, that better mimic human renal physiology. Second, although MSCs exhibit low immunogenicity, their long-term safety profile remains controversial. Pretreatment of tumor cells with a human MSC-CM was sufficient to promote tumor growth, compared to the in vivo co-injection of MSCs in a mouse xenograft model[109]. These findings highlight the importance of long-term tumor surveillance in future clinical trials. Finally, standardized protocols for MSCs isolation, expansion, and delivery are lacking, which affects efficacy outcomes[110]. The translation of preclinical research findings into reliable, efficacious, and secure therapies for patients remains restricted[111], and the best way to perform in vivo implantations, the optimal dosage and duration of treatment, and how to improve the culture conditions of the cells to further enhance the viability and functionality of stem cells in the body is unclear. These issues underscore the need for rigorous trials before making further clinical recommendations.

To enhance the stability and quality of MSCs and MSC-EVs, novel strategies have emerged, including pretreatment technologies or genetic manipulation to optimize the secretory profile of MSCs and integration of MSCs with advanced biomaterials to improve tissue-specific engraftment[112] of synthetic nanoparticles that stabilize MSCs and amplify therapeutic efficacy[113].

In conclusion, advancing the translational applications of MSCs in large animal studies requires the optimization of experimental models, standardization of protocols for manufacturing and characterization, and multidisciplinary collaborations that bridge regenerative medicine and bioengineering.

CONCLUSION

MSCs and their EVs can reduce apoptosis and slow DKD progression. As a novel cell-free therapeutic approach, MSC-EVs can transport miRNAs (such as miR-16-5p and miR-22-3p) to target cells or organs. They can relieve DKD symptoms by regulating apoptosis-related signaling pathways, making them safer and more effective than cell transplantation. However, as a new mode of stem cell application, challenges such as limited large-animal data, controversial long-term safety, and a lack of standardized protocols impede their clinical use. With continuous advancements in technology, MSC-EVs are believed to become an effective strategy for alleviating DKD symptoms in the future.

ACKNOWLEDGEMENTS

We thank our colleagues for suggestions on this manuscript.