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. 2026 Feb 25;27:205. doi: 10.1186/s12882-025-04648-5

Dual role of miR-21-enriched MSC-derived exosomes in diabetic nephropathy: therapeutic potential versus p53-driven pathogenicity

Yian Wang 1,✉, Yongxin Lu 1, Yanhong Zhao 1, Jianqing Xu 1, Lu Gan 1, Chongmeng Yang 1, Chengxian Pi 1, Dechang Chen 1, Xue Liao 1, Yuanhong Mao 1, Yang Sun 1,✉, Zongwu Tong 1,✉
PMCID: PMC13040957  PMID: 41742063

Abstract

Background

Diabetic nephropathy (DN) is a prevalent and devastating complication of diabetes mellitus and is characterized by tubular atrophy, interstitial fibrosis, lipid metabolism dysregulation, and oxidative stress. Mesenchymal stem cell-derived exosomes (MSCs-Exo) have demonstrated promise in attenuating these DN-associated pathologies through multiple mechanisms. However, microRNAs (e.g., miR-21) encapsulated within MSCs-Exo may also have deleterious effects on DN through discrete molecular pathways, and the underlying mechanisms remain unclear.

Objective

This study aimed to elucidate the role of the miR-21/p53 signaling axis in the MSC-Exo-mediated modulation of DN progression and to define the dual role of miR-21 in both therapeutic and pathogenic contexts.

Methods

DN models were established in vitro using high-glucose–treated HK2 cells and in vivo using C57BL/KsJ db/db mice. The effects of MSCs-Exo, an miR-21 mimic, and the p53 activator C16 on cell proliferation, lipid metabolism, fibrosis, and oxidative stress were evaluated. The outcomes were quantified using transmission electron microscopy, qRT‒PCR, Western blotting, ROS fluorescence, hematoxylin and eosin (HE) staining, periodic acid–Schiff (PAS) staining, and Masson’s trichrome staining.

Results

MSCs-Exo significantly restored the proliferation of high-glucose–treated HK2 cells and concomitantly downregulated the expression of miR-21 and p53 and the dysregulation of lipid metabolism, oxidative stress, and fibrosis. In vivo, MSCs-Exo improved glycemic control, lipid profiles, and renal function in diabetic mice but also attenuated tubular atrophy and collagen deposition. However, miR-21–enriched MSCs-Exo markedly activated p53 signaling, exacerbating ferroptosis, lipid accumulation, and fibrosis. Further pharmacologic activation of p53 with C16 worsened these pathological changes, confirming the central role of p53 in miR-21-mediated pathogenic effects.

Conclusion

The MSC-Exo/miR-21/p53 signaling axis plays a critical role in attenuating DN progression. Nonetheless, elevated miR-21 levels in MSCs-Exo activate the p53 pathway and drive ferroptosis, lipid accumulation, and fibrosis, thereby exerting pronounced pathogenic effects. Regulating miR-21 loading in MSCs-Exo may be a key strategy for optimizing their therapeutic efficacy in DN.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12882-025-04648-5.

Keywords: Diabetic nephropathy, miR-21, Ferroptosis, Oxidative stress

Introduction

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes and is among the leading causes of end-stage renal disease (ESRD) worldwide [1, 2]. The pathogenesis and progression of DN involve a complex interplay among multiple pathological processes, including hyperglycemia-induced oxidative stress induced by hyperglycemia, dysregulated lipid metabolism, apoptosis, fibrosis, and chronic inflammation [3, 4]. In recent years, mesenchymal stem cells (MSCs) and their exosomes (MSCs-Exo) have shown considerable promise in the treatment of metabolic diseases and organ injury owing to their immunomodulatory, anti-inflammatory, antioxidant, and antifibrotic properties. Exosomes are nanometer-scale vesicles released by MSCs that contain nucleic acids, proteins, and lipids, and they function as critical mediators of intercellular communication through the transfer of bioactive molecules to recipient cells [5, 6]. Previous studies have demonstrated that MSC-derived exosomes (MSCs-Exo) can improve DN-associated pathologies in both in vivo and in vitro models through the modulation of inflammatory cytokines [7], the cellular microenvironment [8], and fibrosis-related signaling pathways [9].

Despite their therapeutic potential, the biological effects of MSCs-Exo are contingent on the expression profiles of specific microRNA cargos, such as microRNA-21 (miR-21) [10, 11]. miR-21 is a pivotal noncoding RNA that has been implicated in the progression of diverse pathologies and is notably upregulated in high-glucose–induced kidney injury models. It can exert pathogenic effects by modulating the p53 signaling pathway, thereby aggravating fibrosis and oxidative stress [12–15]. Furthermore, p53, a canonical tumor suppressor, plays a crucial role in DN pathobiology. The hyperactivation of p53 exacerbates oxidative stress and dysregulated autophagy and contributes to lipid–metabolic derangements and apoptosis [16–19].

Based on this background, the present study sought to elucidate the mechanisms by which MSCs-Exo modulate the miR-21/p53 signaling axis in DN, with a particular focus on the dual roles of miR-21 in therapeutic versus pathogenic processes. Through a combination of in vitro and in vivo experiments, we systematically evaluated the potential of MSCs-Exo to improve renal function, mitigate fibrosis, and normalize lipid metabolism, thereby establishing a mechanistic basis for the optimization of exosome-based therapies for DN.

Materials and methods

Reagents and cell lines

The human renal proximal tubular epithelial cell line HK2 was obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin‒streptomycin (Beyotime, China) supplemented with 5.5 mmol/L glucose. High-glucose (HG) medium was prepared by adjusting the glucose concentration to 30 mmol/L. Mesenchymal stem cell-derived exosomes (MSCs-Exo) were isolated from human adipose-derived MSC (OriCell, China) culture supernatants by ultracentrifugation and characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).

Experimental grouping

In vitro, HK2 cells were divided into the following groups: untreated control (NC), high glucose (HG), HG + MSCs-Exo, HG + MSCs-Exo-miR-NC, and HG + MSCs-Exo-miR-21 mimic. For in vivo studies, male C57BL/KsJ db/db mice (6 weeks old, SPF grade) were randomly assigned to the same four groups. All the animals were housed and maintained under standard laboratory conditions. To simulate the diabetic microenvironment, HK2 cells (1 × 10⁵ cells/well) were exposed to 30 mmol/L glucose for 48 h, a widely used model dose for diabetic nephropathy in recent SCI literature [20]. To control for hyperosmolarity, the control medium (5.5 mM glucose) was supplemented with equiosmotic mannitol (final concentration of 24.5 mM; Sigma‒Aldrich, Cat# M4125) to match the osmolality of the high-glucose group (30 mM). MSCs-Exo were administered 2 µg/mL (2 mL/well) for 24 h, a dose previously shown to modulate proliferation, fibrosis, and autophagy-related signaling pathways [21]. The p53 activator C16 was used at 20 µM for 20 h based on evidence of its p53 activation and cytotoxic effects in HK2 cells [22, 23]. In vivo, db/db mice were fed a high-fat diet to induce a diabetic state. From week 3, they received tail vein injections of MSCs-Exo (100 µg in 0.2 mL of PBS) three times during the first week and twice weekly for the subsequent three weeks, a dosing regimen that has been validated in chronic kidney disease models to improve pathology and biochemical indices [24]. To assess the role of p53 signaling, mice were also treated with intraperitoneal injections of C16 (10 mg/kg, every other day), a dose shown to exacerbate pathology in diabetic mouse models [25]. All animal experiments in this study were approved by the Animal Ethics Committee of Kunming Medical University (Approval No. kmmu20241426) and were conducted in strict accordance with the guidelines outlined in the BioMed Central Editorial Policies on Animal Research. Male C57BL/KsJ db/db mice (6 weeks old, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal License No. SCXK (Beijing) 2019-0008). The mice were housed under standard laboratory conditions (22 ± 2 °C, 55 ± 5% humidity, 12-hour light/dark cycle) and allowed to acclimate for one week prior to experimentation. To ensure animal welfare, all invasive procedures, including blood collection and tissue harvesting, were performed under inhalation anesthesia using isoflurane (Isoflurane, R510-22, RWD Life Science, Shenzhen, China). Anesthesia was induced with 3–4% isoflurane and maintained at 1.5–2% using an RWD small animal anesthesia system. Anesthetic depth was verified by the absence of a tail-pinch reflex and steady spontaneous respiration. At the end of the experiment, the mice were euthanized under deep anesthesia induced by continuous inhalation of 2.5% isoflurane until loss of the corneal reflex and shallow breathing were observed. Cervical dislocation was then performed to ensure rapid and humane euthanasia.

miR-21 transfection in cells and mice

miR-21 overexpression was achieved using a miR-21 mimic (RiboBio, China). MSCs were transfected using Lipofectamine RNAiMAX (Invitrogen, USA). Cells were seeded at 1 × 10⁵ cells/well, and 50 nM miR-21 mimic was added for 24 h. The supernatants were collected for exosome isolation [26]. For in vivo transfection, MSCs-Exo (100 µg in 0.2 mL of PBS) were injected via the tail vein three times per week for 3 weeks. All procedures strictly followed ethical guidelines for animal studies.

Quantitative Real-Time PCR (qRT‒PCR)

Total RNA from cells and renal tissues was extracted using TRIzol reagent (Invitrogen, USA). Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Takara, Japan). The gene expression levels of miR-21, p53, and other targets were analyzed using SYBR Green Master Mix (Roche, Switzerland) on an ABI 7500 Real-Time PCR system. The 2−ΔΔCt method was used for data analysis. All primers used for qRT-PCR were synthesized by RiboBio (Guangzhou, China). Commercially validated primer sets were used for miR-21 and U6, with the following sequences: miR-21 (forward: 5′-UAGCUUAUCAGACUGAUGUUGA-3′); U6 (forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′). The primer sequences for p53 were forward: 5′-CAGCACATGACGGAGGTTGT-3′ and reverse: 5′-TCATCCAATAATCTCACACGC-3′, yielding a 125-bp amplicon. GAPDH was used as the internal control, with primer sequences forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse: 5′-GGCTGTTGTCAATCTTCTCATGG-3′, producing a 197-bp amplicon.

Western blot analysis

HK-2 cells were washed with ice-cold PBS and lysed on ice for 30 min in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with protease/phosphatase inhibitors; lysates were clarified (12,000 g, 10 min, 4 °C), and protein content was quantified by BCA (Beyotime, China). Equal amounts of protein (20–30 µg) were resolved by 7.5–12% SDS‒PAGE and transferred to 0.2 μm PVDF membranes (Millipore, USA). The membranes were blocked in 5% nonfat milk/TBST (1 h, RT) and incubated overnight at 4 °C with primary antibodies (Abcam, UK) diluted in 5% BSA/TBST as follows: α-SMA (1:1,000; Abcam ab7817), collagen I (1:1,000; Abcam ab34710), GPX4 (1:1,000; Abcam ab125066), TfR1 (1:1,000; Abcam ab84036), FTH1 (1:2,000; Abcam ab65080), and the loading control GAPDH (1:5,000; Abcam ab8245). After the membranes were washed, they were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG-HRP, 1:5,000; Abcam ab205718; and goat anti-mouse IgG-HRP, 1:5,000; Abcam ab205719) for 1 h at RT. Bands were visualized using ECL (Thermo Fisher, USA) and imaged on a chemiluminescence system; molecular-weight markers were run in parallel and annotated on each blot. Densitometry was performed in ImageJ with background subtraction and normalization to GAPDH; for figures, n values and statistical tests are indicated in the legends.

Cell proliferation and lipid accumulation assays

Cell proliferation was assessed with a CCK-8 kit (Dojindo, Japan). Lipid accumulation was evaluated using Oil Red O and Nile Red staining, and the fluorescence signal intensity was observed under a fluorescence microscope.

Detection of biochemical indicators in mice

Mice were kept on a 12:12 h light/dark cycle (ZT0 = lights on), fasted for 6 h with water ad libitum, block randomized by body weight, and assessed under blinding. During ZT2–ZT4, tail-vein blood was collected for fasting blood glucose measurement using an Accu-Chek Guide glucometer (Roche, M07400942001); two readings were averaged with daily QC using vendor solutions.

For serum biochemistry, retro-orbital blood was drawn under isoflurane anesthesia (x − y% in O₂ at a − b L/min), clotted for 30 min at room temperature, and centrifuged (3,000 × g, 10 min, 4 °C). Serum aliquots were stored at − 80 °C and assayed within one freeze–thaw cycle. Blood urea nitrogen (BUN) was measured by a UV microplate kit (Nanjing Jiancheng, C013-3-1) at 340 nm using a standard curve. Total cholesterol (TC) and triglyceride (TG) levels were quantified by enzymatic colorimetry (Nanjing Jiancheng, F002-1-1 and F001-1-1) and reported per-kit calibration [units], read at ~ 500 nm (TC) and ~ 510 nm (TG).

Urine was collected as 24-h metabolic-cage samples (total excretion, mg/24 h) or first-morning spot urine (UP/Cr). Urinary protein (UP) was measured by the pyrogallol red–molybdate method (Quantimetrix QuantTest Red; Fisher Scientific, NC9885166) after sediment removal (3,000 × g, 10 min), read at 600 ± 20 nm and interpolated from a BSA curve. Urinary creatinine was measured by an enzymatic assay (Nanjing Jiancheng, C011-2-1) at 510 nm; UP/Cr is expressed as mg/g. All assays included blanks, standards, and appropriate controls with ≥ 2 technical replicates; n = 6 per group.

Ferroptosis and ROS detection

Immunofluorescence staining was used to detect GPX4 expression as an indicator of ferroptosis. Intracellular reactive oxygen species (ROS) levels were measured using an ROS detection kit (Beyotime, China), and fluorescence was quantified by microscopy.

Histological analysis

Kidney tissues were stained with hematoxylin and eosin (HE), periodic acid–Schiff (PAS), and Masson’s trichrome to evaluate renal tubular injury, basement membrane thickening, and collagen deposition. Histological scoring was performed independently and blindly by two experienced pathologists.

Statistical analysis

All the data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0. Two-group comparisons were analyzed using unpaired Student’s t tests, and comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). A P value of < 0.05 was considered to indicate statistical significance.

Results

MSCs-Exo alleviate diabetic nephropathy by modulating cell proliferation, lipid metabolism, and fibrotic markers

To investigate the therapeutic potential of mesenchymal stem cell-derived exosomes (MSCs-Exo) in diabetic nephropathy (DN), HK2 cells were divided into three groups: untreated control (NC), high glucose (HG), and HG treated with MSCs-Exo (HG + MSCs-Exo). Transmission electron microscopy (TEM) revealed that MSCs-Exo exhibited a typical cup-shaped morphology with a predominant diameter of approximately 100 nm (Fig. 1A). The results of functional assays demonstrated that MSCs-Exo significantly reversed the proliferation of HG-induced HK2 cells (Fig. 1B). qRT‒PCR analysis revealed that compared with HG treatment, MSCs-Exo significantly downregulated miR-21 (Fig. 1C) and p53 expression (Fig. 1D). Biochemical assays revealed marked reductions in triglyceride (TG) and total cholesterol (TC) levels in MSCs-Exo-treated cells (Fig. 1E–F). Western blot analysis revealed that MSCs-Exo significantly reduced the expression of the fibrosis-related proteins α-SMA and collagen I in HG-treated HK2 cells (Fig. 1G), which was further confirmed by densitometric analysis (Fig. 1H–I). These findings suggest that MSCs-Exo exert renoprotective effects on DN by promoting cell proliferation, restoring lipid homeostasis, and inhibiting fibrotic pathways.

Fig. 1.

Fig. 1

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MSCs-Exo alleviate DN-associated pathological changes through multiple regulatory mechanisms. (A) Transmission electron microscopy (TEM) image showing the characteristic cup-shaped morphology and particle size distribution (~ 100 nm) of MSC-derived exosomes (MSCs-Exo). (B) Results of the CCK-8 assay indicating that MSCs-Exo significantly reversed the inhibition of HG-induced proliferation in HK2 cells (n = 3). (C–D) qRT‒PCR results showing that MSCs-Exo reduced the expression of miR-21 (C) and p53 (D) (n = 3). (E–F) Biochemical assays showing that MSCs-Exo significantly decreased total cholesterol (TC) and triglyceride (TG) levels in HG-treated HK2 cells (n = 3). (G) Western blot analysis showing reduced expression of the fibrosis markers α-SMA and collagen I following MSCs-Exo treatment (n = 3). (H–I) Densitometric quantification confirming the significant downregulation of α-SMA (H) and collagen I (I) expression after MSCs-Exo treatment (n = 3). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Overexpression of miR-21 in MSCs-Exo aggravates renal fibrosis via the ferroptosis pathway

To test whether miR-21–dependent ferroptosis contributes to renal fibrosis in DN, we first engineered donor MSCs to overexpress miR-21, isolated their secreted exosomes (MSCs-Exo-miR-21 and MSCs-Exo-miR-NC), and then exposed high-glucose–treated HK-2 cells to these vesicles alongside wild-type MSC-derived exosomes (MSCs-Exo). qRT–PCR of the purified exosomes verified the marked enrichment of miR-21 in MSCs-Exo-miR-21 relative to the levels in both controls (Fig. 2A). CCK-8 assays confirmed decreased cell proliferation upon miR-21 overexpression (Fig. 2B). Western blot analysis revealed the notable downregulation of GPX4 expression and upregulation of TfR1 and FTH1 expression (Fig. 2C), which was further supported by the results of the quantitative analysis (Fig. 2D–F). Immunofluorescence staining revealed significantly decreased GPX4 expression in miR-21-overexpressing cells (Fig. 2G), and fluorescence quantification confirmed these findings (Fig. 2H). These results indicate that miR-21-enriched MSCs-Exo promote ferroptosis, thereby exacerbating renal fibrosis in DN.

Fig. 2.

Fig. 2

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Overexpression of miR-21 in MSCs-Exo regulates the expression of ferroptosis-associated proteins and promotes renal fibrosis in DN. (A) qRT‒PCR of purified exosomes confirming the enrichment of miR-21 in MSCs-Exo-miR-21 versus wild-type MSCs-Exo and MSCs-Exo-miR-NC (n = 3). (B) CCK-8 assay showing reduced HK2 cell proliferation in the HG + MSCs-Exo-miR-21 mimic group (n = 3). (C) Western blot analysis of the ferroptosis-related proteins GPX4, TfR1, and FTH1 (n = 3). (D–F) Quantification of protein levels showing decreased GPX4 (D) and increased TfR1 (E) and FTH1 (F) expression in the miR-21 mimic group (n = 3). (G) Immunofluorescence analysis of GPX4 localization (n = 3). (H) Fluorescence quantification showing reduced GPX4 expression in the miR-21 mimic group (n = 3). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

miR-21-loaded MSCs-Exo exacerbate lipid accumulation in diabetic renal fibrosis

Prior to functional assays, we verified that coculture effectively increased intracellular miR-21 in recipient HK-2 cells: qRT‒PCR revealed significantly higher miR-21 levels in cells exposed to miR-21–enriched MSC exosomes (MSCs-Exo-miR-21) than in cells treated with wild-type MSC exosomes (MSCs-Exo) (Fig. 3A). To investigate the role of miR-21 in lipid accumulation during DN, the levels of intracellular reactive oxygen species (ROS) and the lipid contents were assessed. ROS fluorescence staining revealed a significant increase in the level of ROS in the group treated with the miR-21 mimic compared with that in the group treated with MSCs-Exo alone (Fig. 3B). Moreover, Nile red staining revealed increased intracellular lipid accumulation in miR-21-overexpressing cells (Fig. 3C), with quantification confirming the significantly increased fluorescence intensity in the mimic group (Fig. 3D). Oil Red O staining further corroborated these findings (Fig. 3E). Overall, miR-21-enriched MSCs-Exo significantly aggravated lipid accumulation during diabetic renal fibrosis.

Fig. 3.

Fig. 3

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miR-21-enriched MSCs-Exo exacerbate lipid accumulation and promote diabetic renal fibrosis. (A) qRT‒PCR of recipient HK-2 cells showing increased levels of intracellular miR-21 after coculture with MSCs-Exo-miR-21 compared with wild-type MSCs-Exo (n = 3). (B) ROS fluorescence staining indicating increased ROS levels in the MSCs-Exo-miR-21 group (n = 3). (C) Nile red staining showing increased intracellular lipid accumulation in the miR-21 mimic group (n = 3). (D) Fluorescence quantification confirming the enhanced lipid fluorescence intensity (n = 3). (E) Oil Red O staining showing substantial lipid deposition after miR-21 overexpression (n = 3). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

p53 signaling mediates miR-21-Induced ferroptosis and renal fibrosis in DN

To explore the regulatory role of p53 in miR-21-mediated ferroptosis and fibrosis, HK2 cells were assigned to four groups: HG + MSCs-Exo, HG + MSCs-Exo-miR-NC, HG + MSCs-Exo-miR-21 mimic, and HG + MSCs-Exo-miR-21 mimic + C16 (a p53 activator). Western blot analysis revealed decreased GPX4 expression and increased TfR1 and FTH1 expression in the miR-21 mimic group, indicating ferroptosis activation (Fig. 4A). Upon C16 treatment, p53 was markedly activated, further decreasing GPX4 expression and increasing TfR1 and FTH1 expression. ROS levels were significantly elevated in the mimic group and further enhanced by C16, as shown by fluorescence staining and quantification. Lipid accumulation detected by Oil Red O and Nile Red staining also increased following miR-21 overexpression and was further exacerbated by C16 treatment (Fig. 4C–G). p53 pathway protein analysis revealed that p53 and HO-1 were upregulated, whereas Nrf2 and SLC7A1 were suppressed in the miR-21 mimic group; C16 intensified these effects (Fig. 4H). Collectively, these data indicate that excessive p53 activation aggravates miR-21-induced ferroptosis and fibrosis in DN.

Fig. 4.

Fig. 4

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p53 signaling mediates miR-21-induced ferroptosis and renal fibrosis in DN. (A) Western blots of GPX4, TfR1, and FTH1 in cells exposed to MSCs-Exo, MSCs-Exo-miR-NC, MSCs-Exo-miR-21, or MSCs-Exo-miR-21 plus C16 (p53 activator); miR-21 enrichment decreased GPX4 expression and increased TfR1/FTH1 expression, with C16 expression further increasing these changes(n = 3). (B) ROS fluorescence staining showing elevated ROS levels in the MSCs-Exo-miR-21 group, which further increased with increasing C16 concentration(n = 3). (C) Oil Red O staining indicating enhanced neutral lipid deposition with miR-21 enrichment and additional exacerbation by C16(n = 3). (D) Quantification of ROS fluorescence intensity(n = 3). (E–F) Nile red staining and quantification demonstrating increased intracellular lipid accumulation in the MSCs-Exo-miR-21 and MSCs-Exo-miR-21 + C16 groups(n = 3). (G) Western blots of p53-axis proteins showing upregulated p53 and HO-1 with concomitant downregulation of Nrf2 and SLC7A1 in the MSCs-Exo-miR-21 group; C16 further augments these effects(n = 3). Data are presented as the mean ± SD; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

In vivo validation of the MSCs-Exo-mediated miR-21/p53 axis in DN progression

To further validate the role of the MSCs-Exo/miR-21/p53 axis in DN, the biochemical markers of db/db mice were assessed. Compared with those in the HG group, the fasting blood glucose (FBG), blood urea nitrogen (BUN), and urinary protein (UP) levels in the MSCs-Exo group were significantly lower (Fig. 5A–C). These effects were diminished in the miR-21 mimic group and further deteriorated upon C16 treatment. Lipid profile analysis revealed that MSCs-Exo decreased TC and TG levels (Fig. 5D–E), which increased significantly with increasing miR-21 overexpression and C16 administration. Western blot analysis revealed elevated fibrosis markers (α-SMA and collagen I) in the mimic and mimic + C16 groups (Fig. 5F–I). These results confirm that MSCs-Exo improve DN progression, whereas miR-21 overexpression and p53 activation exacerbate this pathology.

Fig. 5.

Fig. 5

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MSCs-Exo improve the expression of biochemical markers and fibrosis-related proteins in DN through modulation of the miR-21/p53 axis. (A–C) MSCs-Exo significantly reduced fasting blood glucose (FBG), blood urea nitrogen (BUN), and urinary protein (UP); these improvements were reversed by the miR-21 mimic and further worsened by C16 (n = 6). (D–E) Serum TC and TG levels were reduced by MSCs-Exo and aggravated by the miR-21 mimic and C16 treatment (n = 6). (F) Western blot showing decreased GPX4 expression and increased TfR1 and FTH1 expression in the mimic group, which was further intensified by C16 (n = 6). (G–I) Quantitative analysis confirming GPX4 downregulation (G) and TfR1 (H) and FTH1 (I) upregulation (n = 6). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

MSCs-Exo ameliorate lipid accumulation and renal fibrosis via the miR-21/p53 axis

To evaluate histological changes, HE staining revealed that MSCs-Exo markedly improved tubular architecture and reduced interstitial damage. These effects were reversed by miR-21 overexpression and further worsened by C16, as evidenced by tubular atrophy and fibrosis (Fig. 6A). Oil Red O and Nile Red staining revealed reduced renal lipid deposition in the MSCs-Exo group, which increased in the mimic and mimic + C16 groups (Figs. 6B–C), and all histological and lipid deposition indices were quantified (Figs. 6D–F), indicating that p53 activation promotes lipid accumulation and exacerbates lipid metabolism disorders in DN.

Fig. 6.

Fig. 6

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MSCs-Exo reduce lipid accumulation and renal injury via the miR-21/p53 axis. (A) HE staining showing improved renal tubular structure in the MSCs-Exo group; tubular atrophy and interstitial expansion were exacerbated in the mimic and mimic + C16 groups (n = 6). (B) Oil Red O staining showing reduced lipid deposition in the MSCs-Exo group; increased lipid accumulation was observed in the mimic group and worsened in the C16 group (n = 6). (C) Nile red staining confirming increased lipid fluorescence in the mimic and C16 groups, indicating aggravated lipid metabolism disorder due to p53 activation (n = 6). (D–F) Quantification of tubular injury and lipid deposition indices corresponding to panels A–C (n = 6). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

MSCs-Exo suppress fibrotic progression in DN through miR-21/p53 modulation

Western blot results revealed significant reductions in α-SMA and collagen I protein levels in the MSCs-Exo group. In contrast, the expression levels of these fibrosis markers increased in the miR-21 mimic- and C16-treated groups (Fig. 7A). PAS staining revealed that MSCs-Exo alleviated glomerular damage, whereas miR-21 overexpression and p53 activation (C16 treatment) exacerbated these pathological changes (Fig. 7B). Masson’s trichrome staining confirmed that MSCs-Exo reduced collagen deposition, whereas the miR-21 mimic and C16 significantly increased collagen deposition (Fig. 7C). All histological and lipid deposition indices were quantified (Figs. 7D–F), highlighting the pathological role of excessive p53 signaling in renal fibrosis progression in DN.

Fig. 7.

Fig. 7

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MSCs-Exo attenuate renal fibrosis and suppress p53-mediated pathological aggravation in DN. (A) Western blot showing reduced α-SMA and collagen I expression in the MSCs-Exo group; the expression levels of both markers were significantly upregulated in the mimic and C16-treated groups (n = 6). (B) PAS staining showing the amelioration of glomerular damage by MSCs-Exo, which was aggravated by miR-21 overexpression and further worsened by C16 (n = 6). (C) Masson’s trichrome staining confirmed that collagen deposition was reduced by MSCs-Exo and markedly increased in the mimic and C16 groups, supporting the pathogenic role of p53 activation in fibrosis (n = 6). (D–F) Quantification of fibrosis-related indices corresponding to panels B–C (n = 6). All the data are presented as the mean ± SD. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Discussion

This study systematically investigated the role of MSC-derived exosomes (MSCs-Exo) in mitigating diabetic nephropathy (DN) through modulation of the miR-21/p53 signaling axis, thereby highlighting the dual nature of MSCs-Exo in DN. On the one hand, MSCs-Exo confer therapeutic benefits through the attenuation of p53 signaling and downstream pathways involving ferroptosis, lipid dysregulation, and fibrosis. On the other hand, enrichment of miR-21 within exosomes activates the p53 pathway and exacerbates disease progression. Collectively, these findings not only validate the therapeutic potential of MSCs-Exo but also underscore the necessity of optimizing exosomal cargo, thereby informing clinical translation.

MSCs-Exo exhibited robust renoprotective effects in both cellular and murine models. Our results indicate that MSCs-Exo significantly attenuated the high-glucose–induced suppression of proliferation, lipid–metabolic dysregulation, fibrosis, and oxidative stress through the downregulation of miR-21 and p53 expression. These observations are consistent with those of previous studies demonstrating the ability of MSCs-Exo to modulate inflammatory cytokines and antioxidant signaling pathways to mitigate DN pathogenesis [27–30]. Furthermore, our data revealed that MSCs-Exo substantially reduced the expression of fibrosis markers such as α-SMA and collagen I, thereby attenuating tubular atrophy and interstitial expansion. These findings align with reports by Nagaishi et al., who found that MSCs-Exo, together with growth factors and extracellular matrix components, can effectively suppress renal fibrosis [31, 32].

Despite these protective effects, our study also revealed a pronounced pathogenic role for miR-21 in DN progression. The overexpression of miR-21 strongly activated the p53 signaling axis, leading to the downregulation of the expression of GPX4 (a key regulator of ferroptosis), together with increased lipid accumulation and elevated expression levels of fibrosis markers. These findings are in line with those of Aaron D. McClelland and colleagues, who reported that miR-21 is upregulated under hyperglycemic conditions and contributes to tubular cell injury and fibrosis by targeting PTEN and downstream effectors [33]. Additionally, miR-21 targets several antiapoptotic and antifibrotic molecules, including Nrf2 and HO-1, thereby exacerbating pathological damage [34, 35].

Our study further substantiates the dual role of p53 in DN. Under homeostatic conditions, p53 safeguards genomic integrity and enforces cell cycle checkpoints. In contrast, hyperglycemic stress elicits maladaptive p53 activation and aggravates DN pathology by promoting ferroptosis [36, 37], disrupting lipid metabolism [38, 39], and driving profibrotic signaling [40, 41]. Notably, pharmacologic p53 activation with C16 further intensified miR-21 mimic-induced pathological changes, underscoring the central role of p53 in miR-21-driven disease progression. These observations align with the findings of Fuzhe Ma and colleagues, who reported that overactivation of the p53/NRF2 axis exacerbates diabetic renal injury through heightened oxidative stress [42]. Direct evidence has shown that miR-21 regulates p53. Biophysical studies have demonstrated that miR-21-3p directly binds the p53 DNA-binding domain, likely limiting p53–DNA interactions and transcriptional activity [43]. Mechanistically, miR-21 also suppresses p53 pathway activators (i.e., JMY, TP53BP2/ASPP2, and TOPORS), thereby dampening p53 signaling [44]. Notably, canonical miR-21 sites in the human/mouse TP53 3′UTR are not well established; thus, “direct” regulation primarily reflects miR-21-3p–p53 protein interactions plus the targeting of pathway components.

Overall, our findings provide additional evidence for the therapeutic potential of MSCs-Exo in DN, particularly through the modulation of the miR-21/p53 signaling axis. Nonetheless, our data underscore potential risks associated with miR-21-enriched exosomes. Future studies should prioritize rational engineering of MSCs-Exo, for example, by using gene editing to suppress miR-21 or by combining MSCs-Exo therapy with miR-21 inhibitors to maximize efficacy while limiting off-target effects. Moreover, precision delivery platforms leveraging engineered MSCs-Exo could be promising for the treatment of DN and other complex metabolic disorders.

Despite substantial progress in delineating the MSCs-Exo/miR-21/p53 axis in DN, several limitations warrant consideration. First, our study focused on miR-21 and did not systematically profile or functionally investigate other miRNAs present in MSCs-Exo. Second, the animal models used may not fully recapitulate the multifaceted pathology of human DN, underscoring the need for validation in well-designed clinical studies. Finally, the long-term safety, biodistribution, and manufacturing stability of MSCs-Exo-based therapies remain insufficiently characterized and will require further investigation before clinical translation.

Conclusion

This study is the first to systematically elucidate the dual role of MSCs-Exo in DN through miR-21-mediated regulation of the p53 signaling axis. Our findings underscore the importance of rationally engineering exosomal cargo to enhance therapeutic efficacy. In the future, the use of MSCs-Exo combined with gene editing and precision delivery technologies may offer a more effective and safer strategy for the treatment of DN.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (4.5MB, pdf)

Author contributions

Y. Wang collected the data and performed the experiments; Y. Lu and Y. Zhao conducted data analysis and interpretation; L. Gan and J. Xu contributed to exosome extraction and molecular assays; C. Yang and C. Pi participated in animal modeling and sample collection; X. Liao and D. Chen carried out histological staining and quantification; Y. Mao and Y. Zhang assisted in literature review and data organization; Y. Sun contributed to experimental design and methodology guidance; and Z. Tong supervised the entire project, provided critical revisions, and finalized the manuscript.

Funding

Yunnan Provincial Department of Science and Technology Science and Technology Plan-Kunming Medical Joint Special Project (NO. 202501AY070001-055).

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experimental procedures were reviewed and approved by the Animal Ethics Committee of Kunming Medical University (Approval No. kmmu20241426). All experiments were conducted in compliance with institutional guidelines and the BioMed Central Editorial Policies on Animal Research.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Yian Wang, Email: wangyian@kmmu.edu.cn.

Yang Sun, Email: sunyang@kmmu.edu.cn.

Zongwu Tong, Email: tongzongwu@kmmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (4.5MB, pdf)

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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