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. 2024 Nov 19;110(3):165–177. doi: 10.1159/000542609

Hypoxia-Treated Adipose Mesenchymal Stem Cells Derived Exosomes Enhance the Therapeutic Effects on Unilateral Ureteral Obstruction Mice

Chi Zhang a,b,✉, Longjun Cai c, Meimei Ma d, Xiaohui Xie a, Junsheng Wang a, Yuanyuan Zhang e,✉
PMCID: PMC13200829  PMID: 39561719

Abstract

Introduction

The exosomes from adipose-derived mesenchymal stem cells (AMSCs) had therapeutic effects. However, whether the exosomes derived from hypoxia-treated AMSCs could improve renal functions in unilateral ureteral obstruction (UUO) mice remains unclear.

Methods

The exosomes were characterized using a transmission electron microscope and Western blot. Its size distribution was determined using the Zetasizer Nano ZS analysis system. The differentiation ability was assessed by alkaline phosphatase and oil red staining. Consequently, the function of exosomes in inhibiting inflammatory factors was evaluated using an enzyme-linked immunosorbent assay, and apoptosis inhibition was evaluated by Western blot. Finally, the function of exosomes to ameliorate kidney fibrosis was evaluated using quantitative reverse transcription polymerase chain reaction, Western blot, hematoxylin-eosin staining, and Masson staining.

Results

The cultured AMSCs could differentiate into osteoblast and adipocyte. Meanwhile, the cultured AMSCs could effectively secrete the exosomes, which were characterized by around 110 nm diameter and surface marker expression. Exosomes derived from hypoxia-treated AMSCs improved renal functions in UUO mice. The mechanism exploration revealed that exosomes could decrease the TNF-α and IL-6 and inhibit cell apoptosis. Finally, the fibrosis-associated protein was reversed, and the renal dysfunctions were ameliorated in UUO mice.

Conclusion

The exosomes derived from the hypoxia-treated AMSCs have a better effect than those from normal AMSCs in ameliorating renal dysfunctions in UUO mice.

Keywords: Chronic kidney disease, Adipose mesenchymal stem cells, Exosomes, Inflammation, Apoptosis

Introduction

The global incidence of chronic kidney disease (CKD) is about 10%, and approximately, 7 million people proceed to end-stage renal disease [1]. The long duration of the disease and treatment costs have caused a significant financial burden on individuals and society. CKD is a chronic progressive disease caused by various reasons. Therefore, exploring the molecular mechanism of CKD, as well as the effective prevention and therapeutic approaches, is critical for both science and society. Unfortunately, there are no effective medications or treatment approaches to slow or reverse the development of CKD. Hence, novel strategies for CKD therapy are profound. Previous studies have demonstrated that all patients exhibited pathological changes of renal interstitial fibrosis during CKD progression [2]. Renal fibrosis is a complex process involving multiple cells, cytokines, and signaling pathways [3]. Finding novel effective strategies to prevent the progression of renal fibrosis and cure CKD remains a focus for clinical researchers.

Adipose-derived mesenchymal stem cells (AMSCs) are a type of stem cell with strong self-renewal and differentiation potential, which can differentiate into various types of cells, such as osteoblasts, chondrocytes, adipocytes, and so on [4]. In addition, AMSCs also have various biological characteristics, such as immune regulation and anti-inflammatory effects [4]. Recently, an increasing number of studies have shown that AMSCs may have a specific effect on CKD [5]. It showed that transplantation of AMSCs can alleviate renal tissue inflammation, promote renal tissue repair and regeneration, and finally improve renal function. In addition, AMSCs have low immunogenicity and less ethical controversy, making them a safe and feasible treatment approach [5]. However, it is not yet clear how AMSCs improve renal interstitial fibrosis.

Recent research suggested that AMSCs could provide therapeutic benefits through paracrine or autocrine pathways [6]. The therapeutic effect of AMSC is dependent on its ability to regulate angiogenesis, immune regulation, and cell apoptosis. The AMSC secretes bioactive molecules such as cytokines, chemokines, growth factors, and exosomes. These molecules regulate tissue repair, regeneration, and functional recovery. Among these paracrine molecules, exosomes exhibit unique functions in disease diagnosis and treatment, and AMSCs are considered one of the cells with the ability to produce exosomes [7]. It was revealed that hypoxia could upregulate the level of glial-derived neurotrophic factor (GDNF) [8]. Furthermore, it was found that GDNF-modified exosomes derived from AMSCs had a better effect in ameliorating tubule interstitial fibrosis [9]. Moreover, it has been reported that GDNF can enhance the anti-inflammatory effect of therapies based on human AMSCs in renal interstitial fibrosis [10]. Collectively, these results indicate that low oxygen treatment may enhance the therapeutic effect of exosomes, and hypoxia-induced exosomes derived from AMSCs might become a novel therapy method for CKD. This study aimed to investigate the therapeutic effect of the isolated exosomes from AMSCs treated with low oxygen on the unilateral ureteral obstruction (UUO) mouse model.

Materials and Methods

The Culture of AMSCs

AMSCs were purchased from ScienCell Co. (#7510, CA, USA) and were recovered according to the instructions. AMSCs were cultured in a specific MSCM culture medium (Aoinbio, Shanghai, China) for mesenchymal stem cells (MSCs), supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 1% dual antibody (penicillin mixture, Gibco).

Isolation of Exosomes Derived from AMSCs

When the confluence of AMSC cells reached 80%, the original culture medium was replaced with serum-free MSCM culture medium after 1× PBS washing. Hypoxia treatment of AMSCs (H-AMSCs) was conducted in an incubator with 1% O2, 94% N2, and 5% CO2 at 37°C. Normal oxygen treatment of AMSCs (N-AMSCs) was conducted in an incubator with 5% CO2 and 21% O2 at 37°C. After 48 h of incubation, the supernatant was collected, followed by ultracentrifugation at 4°C to isolate the exosomes. The detailed procedure was as follows: Initially, the cells were removed after centrifugation at 200 g for 10 min and at 2,000 g for 20 min. Subsequently, the subcellular components were removed by centrifugation at 16, 000 g for 30 min. Afterward, the supernatant was filtrated through a 0.22 μm filter and centrifuged at 100,000 g for 3 h to obtain exosome particles.

The morphological structure of exosomes was observed using transmission electron microscopy (Beijing Enze Kangtai Biotechnology Co., Ltd., Beijing, China). The size distribution of the exosomes was determined by a dynamic light scattering technique using a Zetasizer Nano ZS analysis system (Zetasizer version 6.12; Malvern Instruments, UK).

Adipogenic Differentiation

AMSCs are cultured in an incubator at 37°C with 5% CO2. When the cell density reached 80–90%, the cells were digested using mild digestive enzymes (NC1004.1, Yocon, Shanghai, China). The digested 4 × 104 cells were seeded into the six-well plate (Corning, NY), which was incubated in an incubator at 37°C with 5% CO2. The medium was changed every 3 days until the cell density reached 100%, which was conducted according to the following: removal of the supernatant followed by the rinsing of pre-warmed PBS 3 times. The cells were inducted for adipogenic differentiation using StemPro Adipogenesis Differentiation Kit (ThermoFisher Scientific) for 21 days. Medium was changed every 3 days. After the induction, the supernatant was discarded, and the cells were rinsed twice with 1× PBS. Then, the cells were fixed with 4% neutral formaldehyde for 30 min, followed by rinsing with 1× PBS twice (Corning) and staining with 1 mL of oil red O dye solution (Sigma, St. Louis, MO, USA) for 30 min. The stained cells were observed under an inverted microscope after discarding the dye and rinsing of 1× PBS.

Osteogenic Differentiation

When the cell confluence of AMSCs reached 80–90%, the cells were digested and seeded into gelatin-coated six-well plates at the density of 2 × 104/cm2, with incubation at 37°C with 5% CO2. The medium was changed every 3 days until the cell density reached 60–70%. After removing the supernatant, the wells were rinsed thrice with pre-warmed 1× PBS. Osteogenic differentiation was achieved by culturing the cells in StemMACS OsteoDiff Media (Miltenyi Biotec) for 10–12 days. Fresh osteoblast induction complete culture medium was replaced every 3 days. After induction, the supernatant of the culture medium was discarded, followed by rinsing with 1× PBS twice and fixation with 4% neutral formaldehyde for 30 min. Afterward, the formaldehyde solution was discarded, followed by rinsing twice with 1× PBS and staining with alkaline phosphatase (ALP, Baiao Laibo, Beijing, China) for 15 min under dark conditions. After removing the staining solution, the cell was rinsed twice with 1× PBS and observed under an inverted microscope.

Chondrogenic Differentiation

Chondrogenic differentiation was initiated in cell dishes at a density of 2 × 105 cells per pellet, using a chondrogenic medium that included 10 ng/mL TGF-β3, 1 mm GlutaMax, 1% Pen-Strep, 4 mm proline, 1 mm sodium pyruvate, 1% ITS Premix supplement, 50 μg/mL ascorbic acid, and 10−4 mM dexamethasone in high-glucose DMEM medium (ThermoFisher Scientific). The chondrogenic medium was replaced every other day for 3 weeks.

Mice Experiment

This study protocol was reviewed and approved by the Ethics Committee of the Affiliated Suqian Hospital of Xuzhou Medical University (approval number L2023058). 6–8 week male C57 mice with an average weight of 15–20 g were purchased from Nanjing Jicui Yaokang Co., Ltd. (Nanjing, China). Only male mice were used to exclude the interference of sex such as hormone in the observed findings. A mouse UUO model was established according to the following protocol. Anesthesia is performed by intraperitoneal injection of 1% pentobarbital sodium solution (Sigma). The abdomen is exposed in a supine position, and after fixation, the surgical area skin is disinfected. The abdominal wall was opened, followed by the exposure of the left ureter. Subsequently, it was carefully peeled off and ligated with a No. 4 silk thread at the upper one-third of the middle. The ureter was sheared off in the middle, and the abdominal cavity was immediately closed. In the sham surgery group, only the ureter was peeled off without ligation, and then the abdominal cavity was directly closed. The UUO group received 150 μL 1× PBS through the tail vein injection; each mouse in the N-AMSCs exosome group and H-AMSCs exosome group was injected with 150 μL exosomes at 2 μg/μL through the tail vein. Fourteen mice were used for each group. Each mouse was euthanized on day 7, and their eyeballs were removed for blood collection. The serum was used for renal function testing. The left kidney was resected, and the renal capsule was peeled off, followed by rinsing with cold 1× PBS. Subsequently, a portion of renal tissue was fixed with 10% neutral formalin (Sigma). The remaining renal tissue is stored at −80°C for future usage.

Serum Biochemistry

The serums extracted from the blood samples of the mice were used to measure the hematological parameters. The serum parameters, such as total protein (TP), blood urea nitrogen (BUN), and creatinine (CRE), were analyzed using an Auto-dry Chemistry Analyzer (Kehua Bioengineering, Shanghai, China).

Histological Analyses and Masson Staining

The kidneys of mice were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) and then embedded in paraffin (Sigma). Hematoxylin and Eosin were conducted as described previously [11]. Briefly, the paraffin-embedded sections were dewaxed in xylene and rehydrated in different ethanol concentrations from 100% to 50% at room temperature. Afterward, the section was stained in hematoxylin (Saiguo Bio, Guangzhou, China) for 3 min, followed by rinsing with running water and dehydration with ethanol for 5 min at room temperature. Finally, the section was stained with eosin and sealed with neutral gum (Absin, Shanghai, China). The histopathological score was semi-quantitatively quantified with a scoring range from Grade 0–5 (0, normal kidney; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; 5, ≥76%), as previously described [12].

For Masson staining, the steps of dewaxing were similar to H&E staining. Afterward, the sections were soaked in potassium dichromate (Sigma) overnight, followed by washing with tap water. Subsequently, the sections were incubated with hematoxylin for 3 min, followed by washing with tap water. Afterward, the sections were stained with ponceau (Merck, Kenilworth, NJ, USA) for 10 min and rinsed with tap water. Finally, the sections were stained with phosphomolybdic acid solution and aniline blue solution (Sigma) for 3 min, respectively. All sections were sealed with neutral gum (Absin) for imaging. All the processes were conducted at room temperature.

Western Blot

Kidney tissues or exosomes were lysed with radioimmunoprecipitation assay buffer (HY-K1001, MCE, Shanghai, China) containing a protease inhibitor cocktail (MCE). Protein was harvested after centrifugation at 14,000 g for 10 min at 4°C. The concentration of protein was determined by the bicinchoninic acid kit (Beyotime). Afterward, the protein was denatured by incubation at 95°C for 5 min. Approximately 20 μg of proteins were loaded into the gels and transferred to the polyvinylidene fluoride membrane. The membranes were blocked in 5% nonfat milk (Beyotime) before incubation with the primary antibodies (Alix [92880, CST, MA, USA], Tsg101 [72312, CST], CD9 [13174, CST], CD63 [52090, CST] for exosomes), and (cleaved-caspase 3 [ab214430, Abcam], COL1A1 [ab34710, Abcam], α-SMA [ab5694, Abcam], E-cadherin [ab76055, Abcam], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH, 2118, CST] for kidney tissues) overnight at 4°C. Subsequently, the washed membranes were incubated with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. The signal detection was performed in the Bio-Rad Imaging System using an enhanced chemiluminescence agent.

Quantitative Reverse Transcription Polymerase Chain Reaction

RNA was isolated from kidney tissues using an RNA isolation kit (Qiagen, Shanghai). The cDNA was synthesized with a cDNA synthesis kit (Thermo Fisher). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the SYBR green PCR master mix (Yeasen, Shanghai, China) with the following primers referring to primer bank: the forward primer GCT​CCT​CTT​AGG​GGC​CAC​T and reverse primer ATT​GGG​GAC​CCT​TAG​GCC​AT of Col1a1 (118131144c1); the forward primer CCC​AGA​CAT​CAG​GGA​GTA​ATG​G and reverse primer TCT​ATC​GGA​TAC​TTC​AGC​GTC​A of α-SMA gene (31982518c1); the forward primer CAG​TTC​CGA​GGT​CTA​CAC​CTT and reverse primer TGA​ATC​GGG​AGT​CTT​CCG​AAA​A of E-cadherin gene (118129809c1); the forward primer AAT​GGA​TTT​GGA​CGC​ATT​GGT and reverse primer TTT​GCA​CTG​GTA​CGT​GTT​GAT of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH primers were designed using Primer Premier 5 software. All the primers were synthesized by GENEWIZ company (Suzhou, Jiangsu, China).

Statistical Analysis

All statistical analysis was performed using SPSS software (version 26.0). The comparisons of data were done by one-way ANOVA, followed by Tukey’s multiple comparisons test. The result was presented with mean ± SD. p with <0.05 indicated the significance of the test. *** means p < 0.001 compared to sham; # means p < 0.05, ## means p < 0.01, ### means p < 0.001 compared to UUO; & means p < 0.05, && means p < 0.01 compared to N-Exo.

Result

Identification of AMSCs and AMSC-Derived Exosomes

The image of AMSC morphology at passage 3 is shown in online supplementary Figure S1a (for all online suppl. material, see https://doi.org/10.1159/000542609). The osteogenic differentiation was evaluated by ALP staining [13]. In our study, after 14 days of osteogenic differentiation, the AMSCs were stained by ALP, and the result showed that the ALP was strongly positive (online suppl. Fig. S1b). Meanwhile, the Oil Red staining was also performed for the adipogenesis, when the AMSCs were induced into adipocytes for 12 days. The capability of AMSCs for adipogenic and chondrogenic differentiation was also successfully identified (online suppl. Fig. S1c, S1d). Subsequently, the exosomes from hypoxia-treated AMSCs were characterized using a transmission electron microscope (Fig. 1a). The size distribution of exosomes was determined by a dynamic light scattering technique using a Zetasizer Nano ZS analysis system, and the average size was around 110 nm (Fig. 1b). Finally, the biomarker of exosomes was evaluated by Western blot. The result showed that the expression of Alix, Tsg101, CD9, and CD63 was similar between non-treated exosomes and hypoxia-treated exosomes (Fig. 1c), as well as the GAPDH expression (online suppl. Fig. S2).

Fig. 1.

Fig. 1.

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Identification of AMSC-derived exosomes. a TEM image of exosomes from hypoxia-treated AMSCs. b Size of the particles in the isolated hypoxia-treated AMSC-derived exosomes obtained through a dynamic light scattering system. c Detection of Alix, Tsg101, CD9, CD63 expressions by Western blot analysis from AMSC-derived exosomes or hypoxia-treated AMSC-derived exosomes.

Exosomes Derived from Hypoxia-Treated AMSCs Ameliorated Renal Dysfunctions in UUO Mice

To evaluate the effect of exosomes derived from hypoxia-treated AMSCs on amelioration of renal function, the index of kidney/weight was assessed. The result showed that the index was increased in UUO mice compared to the control group. However, the index of the mice injected with exosomes from the N-Exo and H-Exo groups was significantly decreased compared to that in UUO mice (Fig. 2a). Importantly, the index of mice from H-Exo groups decreased significantly. Serum BUN was the biomarker of the injured kidney [14]; therefore, the serum BUN was detected. The analysis result showed that the level of serum BUN was elevated in UUO mice compared to control mice. Contrarily, the level of serum BUN significantly decreased in the mice injected with N-Exo exosomes and H-Exo exosomes (Fig. 2b). Meanwhile, the urine TPs were also detected, and the result revealed that urine TP level also dramatically increased in the UUO mice compared to control mice (Fig. 2c). The urine TP from in the mice treated with H-Exo was significantly and greatly decreased compared to that in the mice treated with N-Exo (Fig. 2c). Creatinine clearance serves as a crucial indicator of kidney function and was notably observed to decrease in the UUO mice. When the UUO mice were treated with N-Exo or H-Exo, the CRE clearance significantly increased. Especially, the CRE clearance in the mice from the H-Exo group significantly increased compared to that in the mice from the N-Exo group (Fig. 2d). Taken together, it was concluded that the exosome from the hypoxia-treated AMSCs had a better effect than that from the normoxia-treated AMSCs.

Fig. 2.

Fig. 2.

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Exosomes derived from hypoxia-treated AMSCs ameliorated renal dysfunctions in UUO mice. UUO mice received the injection of exosomes derived from normal AMSCs (N-Exo) or hypoxia-treated AMSCs (H-Exo). Kidney/body weight (a), serum BUN (b), TPs (c), and CRE clearance (d) in urine were measured at 7 days post UUO. 8 mice were used for each group. Data are presented as mean ± SD. ***p < 0.001 compared to sham, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to UUO, &p < 0.05, &&p < 0.01 compared to N-Exo. One-way ANOVA followed Tukey’s multiple comparisons test.

Exosomes from Hypoxia-Treated AMSCs Ameliorated Inflammation in UUO Mice

Inflammation was associated with the occurrence of renal dysfunctions. Therefore, the serum TNF-α and IL-6 were evaluated. The result showed that the serum TNF-α and IL-elevated in UUO mice compared to the normal mice (Fig. 3a, b). When the mice were treated with exosomes derived from normal or hypoxia-treated AMSCs, the level of serum TNF-α and IL-6 significantly decreased with a p value <0.01 (Fig. 3a, b). Especially, the level of these two inflammatory factors in the mice treated with exosomes derived from hypoxia-treated AMSCs was significantly lower than that in the mice treated with exosomes derived from normoxia-treated AMSCs with p value <0.01, which implied that exosomes derived from hypoxia-treated AMSCs had a better suppression effect on TNF-α and IL-6. Meanwhile, the IL-6 and TNF-α from the kidney were also evaluated in different mice. The analysis revealed that both IL-6 and TNF-α from the kidney dramatically increased in the UUO mice, with p value <0.001 (Fig. 3c, d). Similarly, they also decreased in mice treated with exosomes derived from AMSCs (Fig. 3c, d). Additionally, the exosomes derived from hypoxia-treated AMSCs had a better suppression effect on IL-6 and TNF-α from the kidney than those derived from normoxia-treated AMSCs (Fig. 3c, d). Collectively, it was concluded that the exosome derived from hypoxia-treated AMSCs had a better effect on ameliorating inflammation.

Fig. 3.

Fig. 3.

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Exosomes derived from hypoxia-treated AMSCs ameliorated inflammation in UUO mice. UUO mice received the injection of exosomes derived from normal AMSCs (N-Exo) or hypoxia-treated AMSCs (H-Exo). Serum TNF-α (a) and IL-6 (b), IL-6 (c), and TNF-α (d) levels in kidney tissues were measured by ELISA at 7 days post UUO. 8 mice were used for each group. Data are presented as mean ± SD. ***p < 0.001 compared to sham, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to UUO, &p < 0.05, &&p < 0.01, &&&p < 0.001 compared to N-Exo. One-way ANOVA followed Tukey’s multiple comparisons test.

Exosomes Derived from Hypoxia-Treated AMSCs Attenuated Histopathological Change and Fibrosis of the Kidney in UUO Mice

Subsequently, the histopathological variation of the kidney was assessed to evaluate the function of exosomes derived from AMSCs. The result showed that the tissue morphology of kidneys from UUO mice became abnormal. However, the kidney morphology from the mice treated with H-Exo was restored to a normal level compared to the mice treated with N-Exo (Fig. 4a, c). On the other side, the extracellular matrix was stained to evaluate the kidneys in the mice. The result unveiled that the level of extracellular matrix deposition increased in the kidney of UUO mice (Fig. 4b, d). When the mice were treated with N-Exo or H-Exo, the level of extracellular matrix deposition significantly decreased (Fig. 4b, d). Importantly, the level of extracellular matrix deposition in the kidney from the mice treated with H-Exo was lower than that of the mice treated with N-Exo (Fig. 4b, d).

Fig. 4.

Fig. 4.

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Exosomes derived from hypoxia-treated AMSCs ameliorated histopathological change and fibrosis of kidneys in UUO mice. UUO mice received the injection of exosomes derived from normal AMSCs (N-Exo) or hypoxia-treated AMSCs (H-Exo). At 7 days post the surgery, HE staining was used to measure the histopathological change of the kidney (a) and the comparison of the histopathological score (c). Scale bars, 50 μm. Masson's trichrome staining was performed to evaluate the extracellular matrix deposition in the kidney (b) and the percent of positive area was compared among different treated mice (d). Scale bars, 200 μm. Six mice were used for each group. Data are presented as mean ± SD. ***p < 0.001 compared to sham, #p < 0.05, ###p < 0.001 compared to UUO, &&p < 0.01 compared to N-Exo. One-way ANOVA followed Tukey’s multiple comparisons test.

Exosomes Derived from Hypoxia-Treated AMSCs Ameliorated Cell Apoptosis of the Kidney in UUO Mice

The TUNEL analysis showed that the percentage of apoptotic cells was increased in the UUO mice (Fig. 5a, b). When the UUO mice were treated with N-Exo, the percentage of apoptotic cells decreased (Fig. 5a, b). However, the apoptotic cells in UUO mice treated with H-Exo were less than those in the UUO mice treated with N-Exo (Fig. 5a, b). Afterward, the level of cleaved-caspase 3 was also evaluated, and the result showed that the level of cleaved-caspase 3 increased in UUO mice. However, it decreased in the UUO mice treated with N-Exo or H-Exo (Fig. 5c, d). H-Exo had a more severe suppression effect on cleaved-caspase 3 than N-Exo (Fig. 5c, d). Taken together, it was concluded that the exosome functioned in the inhibition of apoptosis, and H-Exo had a stronger suppression effect on apoptosis.

Fig. 5.

Fig. 5.

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Exosomes derived from hypoxia-treated AMSCs ameliorated cell apoptosis of kidneys in UUO mice. UUO mice received the injection of exosomes derived from normal AMSCs (N-Exo) or hypoxia-treated AMSCs (H-Exo). At 7 days post the surgery, TUNEL was performed to evaluate the cell apoptosis in the kidney (a) and the percent of positive area was compared among different treated mice (b). Scale bars = 50 μm. c Western blot was used to measure the protein expressions of cleaved-caspase 3 from the tissue lysis of the kidney. d GAPDH was used as loading control and the expressions were normalized to sham. Six mice were used for each group. Data are presented as mean ± SD. **p < 0.01, ***p < 0.001 compared to sham, #p < 0.05, ##p < 0.01 compared to UUO, &p < 0.05 compared to N-Exo. One-way ANOVA followed Tukey’s multiple comparisons test.

Exosomes Derived from Hypoxia-Treated AMSCs Ameliorated Fibrosis of the Kidney in UUO Mice

α-SMA and collagen-I are reliable indicators of EMT in diverse organs throughout the fibrosis phase [15]. Furthermore, the marker of fibrosis was detected using qRT-PCR in the kidney. The mRNA level of COL1A1 and α-SMA increased in the kidney of UUO mice, while the mRNA level of E-cad decreased in the kidney of UUO mice (Fig. 6a–c). Furthermore, the protein levels of COL1, α-SMA, and E-cad were also evaluated using Western blot. The result showed that COL1 and α-SMA increased in the kidney from UUO mice (Fig. 6d–f, while the E-cad decreased in the kidney from UUO mice (Fig. 6d, g). When the UUO mice were treated with N-Exo, the mRNA and protein levels of COL1 and α-SMA decreased (Fig. 6a, b, d–f), while the E-cad increased (Fig. 6c, d, g). Importantly, the mRNA and protein levels of COL1 and α-SMA in mice treated with H-Exo were lower than that in mice treated with N-Exo (Fig. 6a, b, d–f). On the contrary, the E-cad in mice treated with H-Exo was higher than that in mice treated with N-Exo (Fig. 6c, d, g). Therefore, it was concluded that H-Exo had a better effect on ameliorating histopathological change and fibrosis of the kidney.

Fig. 6.

Fig. 6.

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Exosomes derived from hypoxia-treated AMSCs ameliorated fibrosis of the kidney in UUO mice. UUO mice received the injection of exosomes derived from normal AMSCs (N-Exo) or hypoxia-treated AMSCs (H-Exo). At 7 days post the surgery, the mRNA expressions of COL1A1 (a), α-SMA (b), and E-cad (c) from the kidney were measured by qRT-PCR. d Western blot was used to measure the protein expressions of COL1A1, α-SMA and E-cad from the kidney. e–g The expressions were normalized to sham (e–g). Data are presented as mean ± SD. **p < 0.01 compared to sham, #p < 0.05, ##p < 0.01 compared to UUO, &p < 0.05 compared to N-Exo. One-way ANOVA followed Tukey’s multiple comparisons test.

Discussion

Renal interstitial fibrosis is a dominant histologic feature that predicts renal functional decline in CKD [3, 16]. Although initial deposition of fibrotic matrix after injury may be beneficial for tissue repair, persistent deposition during chronic injury in CKD eventually leads to end-stage renal disease. None of the current therapies effectively resolve renal fibrosis lesions or halt disease progression [17, 18]. Various factors contributed to the process of renal interstitial fibrosis, including an inflammatory reaction, oxidative stress, multiple cytokines, and the transforming growth factor-β1 (TGF-β1) signaling pathway [19, 20]. Renal failure is characterized by inflammation, fibroblast proliferation, and matrix deposition in the renal interstitium. Therefore, it is critical to screen drugs that can combat fibrosis to treat CKD effectively.

Several studies have demonstrated the effectiveness of MSCs in improving renal interstitial fibrosis [21–23]. MSCs are the most widely studied stem cells. However, they lack specific and unique markers when cultured in vitro. MSCs could not only differentiate into adipocytes, osteoblasts, and chondrocytes but also nonmesodermal cells [24]. MSCs have numerous advantages for therapeutic application, such as exerting a wide range of immune regulatory effects through increasing the proportion of regulatory T lymphocytes, reducing the level of pro-inflammatory cytokines, and increasing the level of anti-inflammatory cytokines [25]. Moreover, a previous study showed that MSC-based treatment for diabetic kidney disease downregulated the mRNA levels of IL-1, IL-6, TNF-α, and monocyte chemotactic protein 1 to reduce the infiltration of CD8 T-cells in the kidney. MSCs exist in almost all tissues, except for amniotic fluid and umbilical cord blood [26]. Importantly, adipose tissue has been identified as an important source of MSCs [27]. In 2001, the MSCs were successfully isolated from adipose tissue by Zuk et al. [28]. Human AMSCs can grow adherently with fibroblast-like morphology in vitro culture system [29], the morphology of our AMSCs is consistent with the previously described. Under varying conditions, AMSCs have been shown to differentiate into various mesodermal cells, such as osteocytes, chondrocytes, skeletal muscle cells, liver cells, and cells derived from other germ layers [30, 31], which indicates that AMSCs represent a type of stem cells. In our study, it was demonstrated that AMSCs could be induced into osteoblasts and adipocytes.

AMSCs could produce and release a lot of growth factors, chemokines, and cytokines, which can promote angiogenesis, stimulate extracellular matrix remodeling, and reduce inflammation, apoptosis, and oxidative stress to recover renal fibrosis [4]. Extracellular vehicles of AMSCs play a role in the regulation of fibrosis, which are distinguished and divided into different types according to different diameters, including microvesicles (MVs), exosomes, and apoptotic bodies. Our study revealed that the diameter of exosomes derived from AMSCs was about 110 nm, with the distribution from 30 nm to 200 nm, consistent with other findings [32]. One previous study showed that exosomes derived from MSCs were able to alleviate the deterioration of renal function in the UUO model, and importantly, it was revealed that the therapeutic effect of exosomes was superior to that of MSC alone [33]. Meanwhile, the exosome highly expressed the EV marker proteins, such as HSP70, TSG101, CD81, CD9, and CD63. Consistent with previous studies, the levels of TSG101, Alix, CD9, and CD63 were highly expressed in the isolated exosomes in our study.

Exosomes are widely present in cell culture supernatants and carry a variety of cell-derived proteins, lipids, DNA, mRNA, and miRNAs. It was demonstrated that MVs of MSCs reduced renal tubular cell apoptosis, and significantly reduced damage to renal function [34]. In our analysis, it was found that exosomes derived from AMSCs effectively decreased inflammation factors, such as TNF-α and IL-6, which was consistent with previous studies that MSC could decrease TNF-α and IL-6 to reverse CKD [35]. Importantly, it was found that exosomes derived from hypoxia-treated AMSCs inhibited cell apoptosis. Finally, it revealed that the fibrosis-associated proteins COL1A1 [36], and α-SMA were significantly decreased, while E-cadherin was significantly increased by exosomes derived from AMSCs. Interestingly, it unveiled that the exosomes derived from hypoxia-treated ASMCs had a better effect than that from normal ASMCs to reverse CKD. The reason why exosomes derived from hypoxia-treated ASMCs is unknown.

Exosomes are released when the intracellular endosomes fuse with the plasma membrane [37, 38]. The released exosomes could deliver the contents to trigger the functional response that affects the physiological or pathological status [39]. The contents of exosomes are complex, including proteins, RNAs, and DNAs, which act as messengers for cell communication [40, 41]. The secretion and contents of exosomes can be modulated by other environmental factors, such as oxygen level, type of disease, mechanical stress, or media composition [42]. Several studies have found that hypoxia upregulated the expression of different non-coding RNAs delivered by exosomes. King et al. found high levels of miR-210 in exosomes released by breast cancer cells under hypoxic conditions [43]. Umezu et al. [44] found that hypoxia-resistant multiple myeloma cells produced more exosomes with a significantly higher expression of miR-135b as compared to normoxic cells. Moreover, it was found that miR-26a was increased in BMSC exosomes cultured under hypoxia conditions [45]. Similarly, exosomes derived from bone marrow MSCs cultured under hypoxic conditions functioned as a reservoir for anti-apoptotic miR-19a [46]. Furthermore, evidence showed that miRNA-26a could prevent endothelial cell apoptosis in the context of atherosclerosis [47]. Another study showed that miR-20a-5p was enriched in hypoxia-derived tubular exosomes and protected against acute tubular injury [48]. MiR-20a-5p was also demonstrated to inhibit apoptosis [49]. Therefore, it was speculated that the hypoxia could greatly increase the level of miRNA in the released exosomes, consequently leading to the suppression of cleaved-caspase 3 and finally protecting the kidney cells in CKD mice.

Conclusions

Our analysis conclusively demonstrates that exosomes derived from hypoxia-treated AMSCs significantly reduce the levels of TNF-α and IL-6. Furthermore, they effectively decrease cleaved-caspase 3, inhibiting the apoptosis process and leading to the amelioration of fibrosis progression in UUO mice with CKD. However, this study still has some limitations. First, it is crucial to determine the difference between exosomes from normal AMSCs and exosomes from hypoxic AMSCs. Second, we need to ascertain whether there is a variance in the content of exosomes from different AMSCs. Further investigation is essential to address these issues and gain a clearer understanding of the regulatory mechanism of exosomes in CKD.

Statement of Ethics

This study protocol was reviewed and approved by the Ethics Committee of the Affiliated Suqian Hospital of Xuzhou Medical University (Approval No. L2023058). This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985).

Conflict of Interest Statement

The authors declare no conflicts of interest.

Funding Sources

This study is supported by Development Fund of Suqian Sci&Tech Program (No. K202313); Jiangsu Key Laboratory of Immunity and Metabolism (No. XZSYSKF2022041); Affiliated Hospital of Xuzhou Medical University (No. XYFY202236); Suzhou Sci&Tech Program Medical Application Basic Research (No. SKYD2023093).

Author Contributions

Data curation and validation: Chi Zhang, Longjun Cai, Meimei Ma, Xiaohui Xie, Junsheng Wang, and Yuanyuan Zhang. Funding acquisition and writing – review and editing: Chi Zhang and Yuanyuan Zhang. Resources and supervision: Yuanyuan Zhang. Writing – original draft: Chi Zhang, Longjun Cai, Meimei Ma, Xiaohui Xie, Junsheng Wang, and Yuanyuan Zhang.

Funding Statement

This study is supported by Development Fund of Suqian Sci&Tech Program (No. K202313); Jiangsu Key Laboratory of Immunity and Metabolism (No. XZSYSKF2022041); Affiliated Hospital of Xuzhou Medical University (No. XYFY202236); Suzhou Sci&Tech Program Medical Application Basic Research (No. SKYD2023093).

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Supplementary Material.

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Associated Data

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Supplementary Materials

Supplementary_material-Suppl.1-s1.docx (527.5KB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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