Extracellular Vesicles from Induced Mesenchymal Stem Cells Inhibit Acute Kidney Injury to Chronic Kidney Disease Transition

Hongduk Kim; Sungok Hong; Soo Kim; Tae Min Kim

doi:10.15283/ijsc24127

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Kim, Hong, Kim, and Kim: Extracellular Vesicles from Induced Mesenchymal Stem Cells Inhibit Acute Kidney Injury to Chronic Kidney Disease Transition

Original Article

Int J Stem Cells 2025;18(3):286-300.

Published online: 14 March 2025

DOI: https://doi.org/10.15283/ijsc24127

Extracellular Vesicles from Induced Mesenchymal Stem Cells Inhibit Acute Kidney Injury to Chronic Kidney Disease Transition

Hongduk Kim^1,^*

, Sungok Hong^2,^*

, Soo Kim³

, Tae Min Kim^1,^2,

¹Institutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang, Korea

²Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Korea

³Brexogen Research Center, Brexogen Inc., Seoul, Korea

Correspondence to Tae Min Kim, Graduate School of International Agricultural Technology, Seoul National University, 1447 Pyeongchang-daero, Daehwa-myeon, Pyeongchang 25354, Korea, E-mail: taemin21@snu.ac.kr

Equal contributor:

^* These authors contributed equally to this work.

Received 13 December 2024 Revised 18 January 2025 Accepted 25 February 2025

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Compared with conventional mesenchymal stem cells (MSCs), induced mesenchymal stem cells (iMSCs) from induced pluripotent stem cells are unique cell sources for tissue regeneration. The effect of extracellular vesicles (EVs) secreted from iMSCs on inhibiting acute kidney injury (AKI) to chronic kidney disease (CKD) transition was not reported. In this study, we investigated whether EVs from iMSCs (iMSC-EVs) could inhibit AKI-to-CKD transition. iMSC-EVs exhibited the general characteristics of EVs, such as protein marker expression, morphology, and size. Additionally, iMSC-EVs were detected in renal tissues after intravenous injection. In human renal tubular epithelial cells, the increase in pro-fibrotic gene expression in response to transforming growth factor β1 treatment was decreased by iMSC-EVs. In a mouse model of the AKI-to-CKD transtion induced by folic acid, repeated administration of iMSC-EVs restored renal function at day 14. Specifically, iMSC-EVs reduced interstitial fibrosis, sustained inflammation, various types of cell death, and the number of immune cells infiltrating kidneys. Capillary rarefaction in renal tissue was also reversed by iMSC-EVs. Our results demonstrate that iMSC-EVs reduced interstitial fibrosis, inflammation, and cell death occurring during the CKD transition after AKI. Thus, iMSC-EVs have the potential to block AKI-to-CKD transition.

Keywords: Acute kidney injury, Chronic kidney disease, Extracellular vesicles, Induced mesenchymal stem cell

Introduction

Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles secreted by almost all cell types. Notably, they contain various biomolecules, and their biological characteristics are affected by the cells from which they are derived (1). Thus, extensive research is currently being conducted to develop EV-based therapeutics for many pathological conditions, including cardiovascular, metabolic, inflammatory, immune, degenerative, and fibrotic diseases. Importantly, EVs from mesenchymal stem cells (MSCs) have therapeutic potential for various diseases because of their immune regulatory and tissue regenerative properties (2). However, utilizing MSCs to prepare large quantities of EVs for clinical or industrial applications is challenging. For example, MSCs are inherently heterogeneous, and their proliferative potential diminishes with each division. Additionally, they possess tumorigenic potential, collectively contributing to inconsistent biological profiles (3). In this regard, MSCs produced from induced pluripotent stem cells (induced mesenchymal stem cells, iMSCs) are advantageous over traditional MSCs because single-cell clones can be amplified easily to obtain large numbers of homologous clonally derived iMSCs (4). iMSCs and MSCs share characteristics such as differentiation potential, marker expression, and self-renewal. However, it was demonstrated that the exprssion of genes are distinct between bone marrow-derived MSCs and iMSCs, indicating that iMSCs are unique from conventional MSCs (5). Indeed, several preclinical studies have shown that EVs from iMSCs have therapeutic potential for several diseases, including ischemic osteonecrosis, osteoarthritis, stroke, cancer, and acute kidney injury (AKI) (2).

Chronic kidney disease (CKD) refers to reduced renal function (glomerular filtration rates<60 mL/min per 1.73 m²), pathological albuminuria, or structural abnormalities for over three months. Over 60% of adults aged<80 years have CKD, and its contribution to cardiovascular disease and mortality increases with age (6). It is defined by progressive and irreversible loss of nephrons, diminished renal regeneration capacity, microvascular damage, metabolic abnormalities, oxidative stress, and inflammation, which eventually leads to fibrosis (7). Currently, there are few treatment options for CKD, and no medication has been specifically designed to treat renal fibrosis (8). Therefore, the underlying causes of renal fibrosis must be thoroughly reassessed to identify viable therapeutic targets.

In contrast to CKD, AKI is defined as a sudden and often reversible decline in kidney function that occurs within several days or hours. The AKI-to-CKD transition refers to a process by which an individual who has undergone an episode of AKI eventually progresses to CKD (9). Timely identification and effective treatment of the underlying cause of AKI may lead to full recovery of renal function, thereby preventing the progression to CKD. However, in some cases, AKI can lead to persistent kidney damage and the development of CKD, mostly owing to maladaptive repair or persistent risk factors such as diabetes, high blood pressure, age, or other chronic conditions (10). Coca et al. (11) conducted a meta-analysis and reported that patients with AKI have an increased risk of CKD progression and higher mortality than those without AKI.

In this study, we investigated whether iMSC-EVs exert a protective effect on AKI-to-CKD transition. Cell-based experiments were also conducted using human renal epithelial cells.

Materials and Methods

Cell culture

HK-2 cells (#22190; Korean Cell Line Bank) were cultured in RPMI 1640 (Welgene) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals) and 1% antibiotic-antimycotic (GeneDireX). To examine the effects of iMSC-EVs, HK-2 cells were cultured overnight in low serum (1%) medium overnight and then treated with transforming growth factor-β1 (TGF-β1, 10 ng/mL) in the presence or absence of iMSC-EVs (100 μg/mL) under serum-free conditions for 48 hours.

Isolation of iMSC-EVs

iMSCs were generated from induced pluripotent stem cells (STEMCELL Technologies Inc.) as described in our previous study (12), and iMSCs were established as a working cell bank at the R&D center of Brexogen Inc. iMSCs were cultured in high-glucose DMEM (Welgene) supplemented with 15% FBS and 1% antibiotic-antimycotic solution at 37℃ in 5% CO₂ and 95% humidified air. Cells from passages 6 were collected and seeded at a density of 10,000 cells/cm² in a 150-mm culture dish (SPL). The following day, the medium was replaced with DMEM supplemented with 2% EV-depleted FBS and cultured for an additional 24 hours. EV-depleted FBS was obtained by overnight ultracentrifugation of 20% FBS in DMEM at 100,000 g, as previously described (13). The supernatant was centrifuged for 10 minutes at 300 g and transferred to new tubes, which were then centrifuged for 20 minutes at 2,000 g. This step was followed by another round of centrifugation at 10,000 g for 80 minutes. Subsequently, the supernatant was passed through a 0.2-μm vacuum filter (Merck Millipore). Finally, the EVs were isolated using ultracentrifugation at 100,000 g for 80 minutes, and the pellet was washed again with phosphate buffered saline (PBS). EV pellets were dissolved in EV-free PBS.

Cryo-TEM

A 200-mesh copper grid (MiTeGen) coated with a formvar/carbon film was subjected to hydrophilic treatment. The iMSC-EV suspension (4 μL) was placed on a grid and blotted for 1.5 minutes at 100% humidity and 4℃. iMSC-EVs on the grid were visualized at 36,000× magnification using a Talos L120C FEI TEM (Thermo Fisher Scientific) at 120 kV.

Nanoparticle tracking analysis

The particle size distribution and concentration of iMSC-EVs were measured using a ZetaView Nanoparticle Tracking Analyzer PMX-120 instrument (Particle Metrix). Both types of EVs were diluted in sterile PBS to obtain the optimal volume of nanoparticle tracking analysis (NTA). Measurements were performed at room temperature using a 488-nm laser and a highly sensitive CMOS camera (Particle Metrix) in several repeats. Sample analysis was conducted under the following camera settings and processing conditions: sensitivity 80, shutter 100, two cycles, 11 positions, and NTA software version 8.05.14_SP7.

Fluorescence tracking of iMSC-EVs

iMSC-EVs were stained with 5 μM CellTracker Orange CMTMR tetramethylrhodamine (Thermo Fisher Scientific) for 30 minutes at 37℃. The stained EVs were subsequently isolated via ultracentrifugation at 100,000 g for 80 minutes, after which the pellet was washed with PBS and ultracentrifuged at 100,000 g for 80 minutes (Beckman Coulter). After redissolution in EV-free PBS, 100 μL (400 μg) of iMSC-EVs were injected into the tail vein of mice 6 hours after folic acid (FA) administration. Mice were euthanized by CO₂ asphyxiation after 66 hours. Kidneys were harvested and embedded in O.C.T. Compound (Sakura Finetek) and sectioned in 10-μm increments using a cryotome (Leica). The sections were then stained with phalloidin-iFluor 488 (Abcam) according to the manufacturer’s instructions. Finally, the sections were mounted with a mounting medium containing DAPI (Vector Laboratories) and analyzed under a confocal microscope (Leica TCS SP8 STED; Leica Camera AG).

RNA extraction and real-time quantitative polymerase chain reaction

Total RNA was isolated from HK-2 or renal tissues and cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized from total RNA (1 μg) using a cDNA synthesis kit (PhileKorea Inc.). Quantitative polymerase chain reaction (qPCR) was performed using AccuPower 2X GreenStar qPCR Master Mix (Bioneer) according to the manufacturer’s protocol. mRNA expression was analyzed using real-time qPCR (CFX96 Real-Time PCR System; Bio-Rad). Primer sequences used for amplification are listed in Supplementary Table S1. The expression of target gene mRNA relative to that of Gapdh was quantified using the 2^−ΔΔCt method. Each experiment was performed in triplicate.

Animal experiments

All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (14). The design, method, and data presentation of animal studies were reported as described in the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (15). All animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (No. SNU-230116-4). Male C57/BL6 mice weighing 21∼22 g were obtained from Koatech and housed at 22℃∼23℃, and 40%∼60% humidity, with a 12/12-hour light/dark cycle under specific pathogen-free conditions. After one week acclimatization period, CKD transition was induced by a single intraperitoneal injection of 200 mg/kg FA in 300 mM sodium bicarbonate. This model is simple (i.e., single administration of FA), and recapitulates most of the interstitial fibrosis found during CKD transition (16, 17). A total fourteen animals were used. To assess their treatment effect, FA-treated animals (N=10) were randomized, and iMSC-EVs (400 μg in 100μL of PBS) or PBS were intravascularly administered on days 1, 4, 7, 10, and 13 after FA injection. To minimize the variance, animals were administered with iMSC-EVs (N=5, single cage) first and then next with saline (N=5, single cage). On day 14, mice were euthanized by CO₂ asphyxiation. Animals that received solvent (300 mM sodium bicarbonate) at day 0 (N=4) were used as normal group. No adverse events was observed in all animals. Data from all animals was included.

Serum biochemistry

Blood was collected via cardiac puncture and incubated at room temperature (23.0℃∼25.2℃) for 20 minutes in a BD Microtainer Serum Separator Tube (BD Biosciences). After centrifugation at 3,000 g for 20 minutes, blood urea nitrogen (BUN) and creatinine levels in the serum samples were measured using a Catalyst Dx Chemistry Analyzer (IDEXX Laboratories, Inc.).

Immunohistochemistry

Formalin-fixed, paraffin-embedded slides were deparaffinized in xylene and rehydrated in descending order of ethanol (100% to 70%). For antigen retrieval, Tris-EDTA (pH 9.0; Abcam; for CD31) or citrate buffer (pH 6.0; Abcam) was used according to the manufacturer’s instructions. The sections were incubated with primary antibodies (Supplementary Table S2) overnight at 4℃. For chromogenic detection, an UltraVision LP Detection System HRP DAB kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. After washing in PBST four times, reactivity was validated using a mouse/rabbit-specific HRP/DAB IHC Detection Kit (micro-polymer, ab236466; Abcam). The slides were washed four times with distilled water and counterstained with Mayer’s hematoxylin (4science) for 1.5 minutes at room temperature. After washing under running tap water, the slides were dehydrated in ascending order of ethanol (70%∼100%). Representative images were obtained using an Olympus BX43 light microscope (Olympus). After staining with CD31 or cleaved caspase 3 antibodies, the positively stained cells were quantified by calculating the mean number of positive cells from 10 non-overlapping fields per slide at 400× magnification.

Immunoblotting

Renal tissues were collected from AKI mice and washed with ice-cold PBS. After tissues were lysed with RIPA buffer (Biosolution) containing a protease inhibitor cocktail (Roche) for 20 minutes and centrifuged at 12,000 g for 15 minutes at 4℃, the supernatant was collected. Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat dry milk or 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST) and incubated overnight at 4℃ with primary antibodies. Detailed information on the primary antibodies used in this study is provided in Supplementary Table S3. Membranes were then washed thrice with TBST for 15 minutes each and incubated for 1 hour at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibodies (Supplementary Table S3). The immunoreactivity of the antibodies was examined using an enhanced chemiluminescence kit (Thermo Fisher Scientific), and electrochemical images were obtained with an Azure Imaging System (Azure Biosystems). Relative band densities were quantified using ImageJ software (version 1.50; National Institutes of Health).

Statistical analysis

Statistical analyses were performed using analysis of variance followed by Tukey’s multiple comparison test. All analyses were performed with GraphPad Prism software version 9.0. Differences at p<0.05 were considered significant.

Results

Characterization of iMSC-EVs

Western blot analysis revealed that the iMSC-EVs were positive for markers typical of EVs (CD9, CD91, and TSG101). These markers were also detected in iMSCs (Fig. 1A). The expression of calnexin, a protein found in cellular organelles, was observed only in iMSCs (Fig. 1A). The average size of the iMSC-EVs was approximately 125 nm, as determined by NTA and cryo-TEM (Fig. 1B, 1C). To confirm whether iMSC-EVs localized to the kidney, fluorescently labeled EVs were intravascularly injected into mice 6 hours after FA injection, and localization was examined after another 66 hours. iMSC-EVs were detected in renal tissues (Fig. 1D). Next, we examined whether iMSC-EVs could reduce the mRNA expression of genes involved in fibrotic changes induced by TGF-β1 in renal proximal tubular epithelial cells (TECs). We observed that the expression of COL1A1, SLUG, FIBRONECTIN, α-SMA, and VIMENTIN was increased by TGF-β1, and that all were reduced by iMSC-EVs (Col1a1, p<0.0001; Slug, p<0.05; Fibronectin, p<0.05; α-SMA, p<0.01; Vimentin, p<0.05; vs. TGF-β1-treated cells; Fig. 1E).

Effect of iMSC-EVs on blocking AKI-to-CKD transition

On day 14 of FA administration, significantly reduced renal function was observed, as indicated by increased levels of BUN (p<0.01) and creatinine (p<0.0001). No changes in body or kidney weight were noted in FA-treated mice compared with that in untreated mice. In contrast, iMSC-EVs markedly enhanced renal function (Fig. 2A) (p<0.01 vs. vehicle-treated mice). Masson’s trichrome staining revealed that the kidneys of FA-treated mice strongly stained with collagen in the interstitium, which was reduced in animals that received iMSC-EVs. Similar findings were observed in tissues with Sirius Red staining (Fig. 2B). The kidneys of FA-treated mice had enlarged tubules, most likely owing to the retention of urine (as shown by the hyaline casts; Fig. 2C). Other types of damage included proliferative lesions, disorganized tubules, loss of tubular cells, and cell debris in the lumen. These changes were ameliorated by iMSC-EVs. No change in mesangial proliferation or basement membrane thickness was observed. The number of interstitial vessels, as indicated by CD-31-positive cells, was reduced in FA-treated mice (p<0.001 vs. normal mice). However, capillary density was increased by iMSC-EVs (p<0.05 vs. animals receiving vehicle; Fig. 2D).

Anti-fibrotic function of iMSC-EVs

Immunostaining revealed that the renal expression of α-SMA, VIMENTIN, FIBRONECTIN, and COL1A1 was enhanced in FA-treated mice. In contrast, their expression levels were reduced by iMSC-EVs (Fig. 3A). Immunoblotting further confirmed that the increase in these protein markers was repressed by iMSC-EVs compared with that in the vehicle-treated mice (α-SMA, p<0.05; COL1A1, p<0.001; phosphorylated SMAD2/3, p<0.05; VIMENTIN, p<0.05; Fig. 3B).

Reduction in renal cell death by iMSC-EVs

Next, we investigated whether iMSC-EVs could reduce cell death in the kidneys of FA-treated mice. The kidneys of FA-treated mice exhibited increased expression of BAX, which was reduced by iMSC-EVs (Fig. 4A). Moreover, the number of cells expressing cleaved caspase 3, another marker of apoptosis, increased in FA-treated mice and was reduced by iMSC-EVs (p<0.001 vs. vehicle-treated mice) (Fig. 4A, 4B). Immunoblotting revealed that the expression of markers of apoptosis (BAX) and necroptosis (MLKL, phosphorylated RIP3 [p-RIP3]) was increased by FA, which was then reduced by iMSC-EVs (BAX, p<0.001; MLKL, p<0.05; p-RIP3, p<0.05; vs. vehicle-treated mice). Similarly, changes in the expression of ferroptosis markers (FTH1 and GPX4) were observed in FA-treated mice compared with normal tissue; restoration of GPX4 expression by iMSC-EVs was observed, whereas no change was observed in FTH1 (Fig. 4C, 4D).

Anti-inflammatory function of iMSC-EVs

Immunohistochemical analysis indicated that renal expression of NGAL and TNF-α was augmented in FA-treated mice, whereas their expression was reduced in mice that received iMSC-EVs (Fig. 5A). Real-time qPCR analysis revealed that the mRNA expression of NGAL, TNF-α, TIMP-1, VCAM-1, and ICAM-1 was increased in FA-treated mice, and that this increase was reversed by iMSC-EVs (Fig. 5B). Western blotting revealed that, in iMSC-EV-treated mice, the expression of NGAL and ICAM-1 was lower than that in the vehicle group (NGAL, p<0.05; ICAM-1, p<0.05; Fig. 5C). Additionally, we observed that the expression of PCNA and CDC2, which are likely expressed in response to inflammation (18), was increased by FA treatment. However, iMSC-EVs reduced their expression (PCNA, p<0.05; CDC2, p<0.05; vs. vehicle-treated mice; Supplementary Fig. S1).

iMSC-EVs block immune cell infiltration

A higher number of cells expressing CD45 (pan-leukocytes), F4/80 (macrophages), CD11c (M1 macrophages), and CD206 (M2 macrophages) were observed in the kidneys of FA-treated mice (Fig. 6A, 6B) compared with those of control animals. Immunoblot analysis indicated that the expression of these proteins decreased in response to iMSC-EVs (CD45, p<0.01; F4/80, p<0.05; CD11c, p<0.01; CD206, p<0.01; vs. vehicle-treated mice; Fig. 6B). The number of CD3-or Ly6G-positive cells increased in the kidneys of FA-treated mice (Fig. 6C), and iMSC-EVs led to a reduction in the number of CD3-expressing cells compared with that in vehicle-treated mice (p<0.01 vs. vehicle-treated mice). No significant change in Ly6G-positive cells was observed in response to the iMSC-EVs, although a slight reduction was noted (Fig. 6C).

Discussion

Maladaptive repair after AKI can reportedly contribute to the progression of CKD and severe or repeated episodes of AKI are associated with CKD development (19). However, no therapies are currently available to block the progression of CKD transition after AKI. In the present study, we evaluated whether repeated administration of iMSC-EVs could repress the fibrotic transition of kidneys induced by FA treatment. We first observed that iMSC-EVs markedly reduced the mRNA expression of pro-fibrotic genes in HK-2 cells undergoing the mesenchymal transition by TGF-β1. Animal experiments demonstrated that renal function, collagen deposition, and tissue structure were enhanced by iMSC-EVs 14 days after FA administration. Consequently, immunohistochemical and immunoblot analyses were conducted on renal tissues to confirm the observed renoprotective functions of iMSC-EVs. iMSC-EVs blocked fibrosis, inflammation, cell death, and reduced the number of immune cells infiltrating the kidneys. iMSC-EVs also increase renal capillary density. However, to determine the mechanism of iMSC-EVs in inhibiting CKD transition, it is needed to perform studies with varying treatment regime (repetition and dosage) followed by high-throughput transcriptomic analysis (e.g., single cell RNA sequencing from specific part of the tissue) and bioinformatic analysis (20). Proteome profiling as well as small RNA (miRNA) sequencing of iMSC-EVs followed by target gene function analysis can also provide novel insights in understanding the function of iMSC-EVs (21).

Various animal models of the AKI-to-CKD transition, such as those induced by unilateral ureteral obstruction (UUO), 5/6 nephrectomy, cisplatin, and FA, are commonly used (22). One advantage of the FA-induced model is that the transition can be induced by a single intraperitoneal injection of FA, resulting in less deleterious outcomes in other organs, unlike that induced by cisplatin, which requires multiple administrations that may contribute to pathologies including DNA damage (23, 24). In addition, FA is a vitamin B that is necessary for normal cell growth and function at low doses and is generally recognized as non-toxic. However, it should be noted that this model has limitations including low possibility of using high dose of FA in clincal setting, and unclear molecular and biochemical mechanisms underlying fibrotic transition (17). One possible mechanism of FA on renal injury can be explained by its metabolism in TECs. With high-dose treatment, FA is converted to tetrahydroxy folate and freely filtered by the glomeruli, which are subsequently reabsorbed into proximal TECs by folate receptor 1 (20). In TECs, folate is enclosed in endosomal vesicles that fuse with the membranes of other organelles, contributing to functional and structural impairments. Approximately 40% of folate accumulates in mitochondria, inducing mitochondrial oxidative stress and inflammation and leading to renal damage and dysfunction (25). Thus, iMSC-EVs may inhibit the oxidative stress induced by FA in TECs, subsequently preventing inflammation and the fibrotic transition of TECs. Further detailed mechanistic studies (e.g., single-cell transcriptomic studies at different time points) are required to better identify the cells affected by iMSC-EVs.

Multiple factors contribute to the development of interstitial fibrosis after AKI. The acute consequences of AKI may begin with maladaptive repair of tubular cells and inflammation (26). In FA-induced AKI, folate causes cell death in TECs through the secretion of various damage-associated molecular patterns (DAMPs). Consequently, DAMPs interact with pattern recognition receptors on TECs, propagating the damage and leading to the production of pro-inflammatory cytokines and chemokines by TECs (27). Local inflammation is further aggravated by the infiltration of immune cells, particularly macrophages, which induces further damage to TEC. Exosomes from cultured TECs were recently observed to undergo an epithelial-to-mesenchymal transition induced M1 polarization of macrophages, which promoted inflammation (28). This study suggests the possibility of indirect intrarenal communication (29). However, the mechanisms by which systemically injected iMSC-EVs reach the tubular compartment, separate from the vascular network of peritubular capillaries or glomeruli, remain unclear. To explore this, Tang et al. (30) demonstrated that mouse red blood cell-derived EVs, engineered to express the Kim-1-binding LTH peptide, could deliver siRNAs for p65 and Snai1 to ischemia/reperfusion (IR)- or UUO-induced fibrotic changes. This result is meaningful in that they specifically targeted p65 and Snai1, which are important molecules in the pro-inflammatory and pro-fibrotic states of TECs, respectively (31), and that they simply treated red blood cells with calcium ionophores, which can provoke EV secretion (32).

Various types of cell death occur during renal injury (33). Martin-Sanchez et al. (34) reported that inhibition of ferroptosis led to the recovery of renal function and reduced tissue damage, oxidative stress, inflammation, and tubular cell death; however, these findings were not observed upon inhibition of necroptosis or apoptosis. Notably, blocking ferroptosis prevented upregulation of MLKL and RIP3 expression, indicating that ferroptosis augments necroptosis. These findings suggest that ferroptosis, compared with necroptosis or apoptosis, is more critical for AKI at 48 hours in FA-induced AKI mice. However, the relationship between renal cell death, maladaptive tubule repair, persistent inflammation during the AKI-to-CKD transition remains largely unexplored. Our data suggest that inflammation, fibrosis, and various types of cell death, including apoptosis (BAX and cleaved caspase 3), necroptosis (MLKL and RIP3), and ferroptosis (GPX4), were actively occurring on day 14 of FA treatment, all of which were reduced by repeated administration of iMSC-EVs. Thus, it would be needed to determine the mechanism underlying how iMSC-EVs inhibit ferroptosis in FA-mediated renal injury (33, 34).

Macrophages play a critical role in renal fibrosis following AKI. They are functionally classified into classically activated M1 or alternatively activated M2 types, which are associated with Th1- or Th2-like immune responses, respectively (35). In the present study, we observed that infiltration of both M1 and M2 macrophages increased and that all were decreased by iMSC-EVs. The result of M1 macrophage is consistent with that of our previous study, which showed that iMSC-EVs reduced TNF-α, IL-1β, and phosphorylated p65 levels in M1-induced THP1 macrophages (36). Similarly, Lu et al. (37) observed that the polarization of both M1 and M2 macrophages were inhibited by exosomes from bone marrow-derived MSCs through activation of prostaglandin receptor 2 using a mice model of UUO-induced interstitial fibrosis. Concordantly, Clements et al. (38) used a renal bilateral IR injury model and reported that Ly6C^high monocytes increased in injured kidneys and could be further classified into three subsets: pro-inflammatory CD11b^＋/Ly6C^high cells being the most dominant at the onset of renal injury, CD11b^＋/Ly6C^int monocytes increasing in the renal repair phase, which were identified using PCNA staining, and pro-fibrotic CD11b^＋/Ly6C^low monocytes increasing in the late phase (for up to day 35 of IR injury). This report is meaningful that late phase macrophages could be classified into reparative and pro-fibrotic types. A comprehensive investigation of the mechanisms by which iMSC-EVs control macrophage polarization would help identify key therapeutic targets to inhibit AKI-to-CKD transition.

The delivery of EVs to renal endothelial cells offers an important strategy for the treatment of renal diseases because damaged endothelium can cause renal function to deteriorate through the loss of capillaries and adherence of inflammatory cells (39). However, the mechanisms by which EVs reach these vessels remain unexplored. To facilitate EV targeting of endothelial cells, Zhang et al. (40) demonstrated that placenta-derived EVs engineered to express P-selectin-binding peptide showed better functionality in reducing inflammation, loss of capillaries, and maladaptive repair of tubules than those treated with non-engineered EVs on day 3 of IR injury. Furthermore, these engineered EVs showed enhanced anti-fibrotic functioning relative to non-engineered EVs on day 28 of injury, indicating that a single administration of EVs targeting the renal endothelium is sufficient to inhibit fibrosis (40). Combined with drug loading, targeting TECs or the endothelium would be next platform to enhance the therapeutic efficacy of EVs in renal diseases.

In conclusion, our findings provide evidence that iMSC-EVs exert a protective effect during AKI-to-CKD transition by reducing pathological features including interstitial fibrosis, inflammation, and cell death. These results underscore the therapeutic potential of iMSC-EVs in mitigating CKD transition from AKI.

Supplementary Materials

Supplementary data including three tables and one figure can be found with this article online at https://doi.org/10.15283/ijsc24127

ijsc-18-3-286-supple.pdf

Acknowledgments

We thank the Designed Animal Research Center, Institute of Green Bioscience and Technology, Seoul National University, for their support in animal care and tissue preparation.

Notes

Potential Conflict of Interest

There is no potential conflict of interest to declare.

Authors’ Contribution

Conceptualization: HK, SK, TMK. Data curation: HK, SH, TMK. Formal analsys: HK, SK, TMK. Funding acquisition: SK, TMK. Investigation: HK, SH, TMK. Methodology: HK, TMK. Project administration: TMK. Resources: SK, TMK. Supervision: TMK. Validation: HK, SK, TMK. Writing – original draft: HK, SH, TMK. Writing – review and editing: HK, TMK.

Funding

This work was supported by the National Research Foundation of Korea through a grant funded by the Korean Government (MSIT) (RS-2024-00336067).

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Fig. 1

Characterization of induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs). (A) Immunoblot analyses of markers for EVs. The expression of markers for EVs (CD9, CD81, and TSG101) or organelles (calnexin) was analyzed in iMSCs and iMSC-EVs. (B) Size distribution of iMSC-EVs. Sizes were measured using a nanotracking particle analyzer. (C) The morphology of iMSC-EVs. Cryo-TEM was used for imaging. Scale bar=200 nm. (D) Fluorescence detection of iMSC-EVs in kidneys. CMTMR-stained iMSC-EVs (400 μg) were intravascularly injected into mice 6 hours after folic acid administration. After 66 hours, the kidneys were harvested, and the presence of iMSC-EVs was detected under laser confocal microscopy. Sections were counterstained with Phalloidin (green) and DAPI (blue). Control mice received phosphate buffered saline. Scale bar=50 μm. (E) The effect of iMSC-EVs on the mRNA expression of fibrotic markers in renal proximal tubular epithelial cells. HK2 cells were treated with TGF-β1 (10 ng/mL) in the presence or absence of iMSC-EVs (100 μg/mL) under serum-free conditions for 48 hours. N=3. Data are presented as mean±SD. *p<0.05, **p<0.01, and ****p<0.0001. CL: cell lysate.

Fig. 2

Induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs) reduce the acute kidney injury-to-chronic kidney disease transition. (A) Concentrations of blood urea nitrogen, serum creatinine, body weights, and kidney weights. N=4∼5. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (B) Microscopic images of kidney sections stained with Masson’s trichrome or Sirius Red. Magnification: 200×. Scale bar=100 μm. (C) Images of kidney sections stained with PAS. Magnification: 200×. Scale bar=100 μm. (D) Immunohistochemical staining of renal vessels by CD31 antibody. Magnification: 400×. Scale bar=100 μm. *p<0.05 and ***p<0.001.

Fig. 3

Alleviation of fibrotic changes by induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs) in the kidneys of an acute kidney injury-to-chronic kidney disease model. (A) Immunohistochemical detection of α-SMA, Vimentin, Fibronectin, and Collagen 1. Scale bar=100 μm. Magnification: 200×. (B) The protein expression of α-SMA, Collagen 1, phosphorylated SMAD, and Vimentin was detected using immunoblotting. Each lane represents a sample from one animal. After antibodies corresponding to each protein were used for immunoblotting, the expression of each protein was normalized against that of β-actin and quantified using ImageJ software. N=4∼5. Data are presented as mean±SD. *p<0.05 and ***p<0.001. N represents normal animals that had not undergone injury.

Fig. 4

Decrease of renal cell death by induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs) during the acute kidney injury-to-chronic kidney disease transition. (A) Immunohistochemical detection of BAX and cleaved caspase expression in kidneys. Scale bar=100 μm. Magnification: 200× (upper) and 400× (bottom). (B) Comparison of the number of cells positive for cleaved caspase 3. N=4∼5. *p<0.05, ***p<0.001, and ****p<0.0001. (C, D) Immunoblot analysis of the markers for apoptosis (BAX), necroptosis (MLKL, RIP3), and ferroptosis (FTH1, GPX4) were analyzed in injured kidneys. Each lane represents lysate from a single animal. The expression level of each protein was normalized against that of β-actin (Fig. 3B) and then quantified using ImageJ software; N=4∼5. Data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Fig. 5

Induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs) alleviate inflammation in the kidneys of mice undergoing the acute kidney injury-to-chronic kidney disease transition. (A) Immunohistochemical detection of NGAL and TNF-α in kidney tissues. Scale bar=100 μm. (B) Analysis of mRNA expression by real-time quantitative polymerase chain reaction. Data are presented as mean±SD. *p<0.05, **p<0.01, and ***p<0.005. (C) Immunoblot analysis of inflammatory genes (NGAL, TNF-α, ICAM-1) was conducted in injured kidneys. Each lane represents lysate from a single animal. The expression of each protein was normalized against that of β-actin shown in Fig. 3B and then quantified using ImageJ software; N=4∼5. Data are presented as mean±SD. *p<0.05 and **p<0.01.

Fig. 6

Inhibition of immune cell infiltration by induced mesenchymal stem cell-extracellular vesicles (iMSC-EVs) in the kidneys of mice undergoing the acute kidney injury-to-chronic kidney disease transition. (A) Immunohistochemical detection of CD45, F4/80, CD11c, and CD206 in kidney tissues. Magnifications: 400× (CD45 and F4/80) and 200× (CD11c and CD206). Scale bar=100 μm. (B) Immunoblot detection of macrophage markers in kidneys. Each lane represents lysate from a single animal. The expression of each protein was normalized against that of β-actin (Fig. 3B) and then quantified using ImageJ software. N=4∼5. Data are presented as mean±SD. *p<0.05 and **p<0.01. (C) Immunohistochemical detection of CD3- or Ly6G-expressing cells in kidney tissues. Scale bar=100 μm. N=4∼5. Data are presented as mean±SD. *p<0.05, **p<0.01, and ****p<0.0001.

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