Introduction
Diabetic nephropathy (DN) is a common complication of diabetes and is recognized as the leading cause of end-stage kidney disease [
1]. Up to one-third of patients with diabetes are affected by impaired renal function, leading to poor prognosis and a heavy social and economic burden over time [
2].
Mesenchymal stem cells (MSCs) have received increasing attention in recent years for their promising anti-inflammation and immunomodulatory functions [
3,
4]. Adipose-derived stem cells (ADSCs), which can be easily harvested from various tissues, are multipotent and can secrete bioactive factors, such as proteins and RNA [
5]. These factors are often secreted in the form of extracellular vesicles—including exosomes (Exo) which are nanoscale vesicle 30–150 nm in size—and transported to various parts of the body through autocrine, endocrine, and paracrine pathways to play a role in regulating tissue homeostasis [
6,
7]. Consequently, exosomes can serve as biomarkers and therapeutics for various kidney disorders [
8,
9]. ADSCs-Exo have become a subject of interest in DN therapy in recent years [
10]. We also found that ADSCs-Exo ameliorated the HG-stimulated podocytes and DN albuminuria progression [
11]. However, the specific mechanisms remain unclear and require further research.
Current researches have provided evidence that inflammatory responses and oxidative stress play crucial roles in DN progression [
12‐
14], leading to increased proteinuria, glomerular endothelial cell damage, and renal fibrosis, triggering the onset of DN [
15]. Interestingly, MSC-derived exosomes have been found to exert anti-inflammatory and anti-oxidative functions in a variety of diseases. [
16,
17].
The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a classical antioxidant stress pathway that mediates the transcriptional regulation of various antioxidant factors [
18,
19]. Physiologically, Nrf2 can be trapped by Keap1 and degraded through the proteasome pathway to maintain a low levels [
20]. Under stress, Nrf2 dissociates from Keap1 and is transported to the nucleus to specifically regulate ARE transcription [
21]. As an anti-inflammatory, proliferative, angiogenic, and cytoprotective enzyme, Heme oxygenase-1 (HO-1) is an influential downstream factor of this pathway, exerting antioxidant effects [
22] which can heal diabetic foot wounds [
23].This pathway has a potential role in reversing high glucose (HG)-induced podocyte injury, although the specific mechanism is not fully understood.
Therefore, we investigated the mechanism underlying ADSC-Exo function in DN progression. We demonstrated that ADSCs-Exos can reduce oxidative stress and inflammation in podocytes via the Keap1/Nrf2/ARE pathway. This lays the groundwork for further clinical applications of ADSC-Exo.
Materials and methods
Exosome isolation and identification
Exosome extraction and identification were performed as previously described [
11,
24]. The exosomes used in this study were extracted by our research group and frozen in liquid nitrogen for long-term use.
Cell culture and treatment
MPC5 mouse podocytes were obtained from icell Bioscience (iCell-m081, Shanghai, China). The cells were seeded into culture flaps coated with type I collagen (Gibco,17100-017) and amplified in RPMI-1640 medium (Hyclone, SH30809.01b) with 20 U/mL IFN-γ (GMP-TL105) and 10% fetal bovine serum (Gibco, San Diego, CA, USA) at 33 °C. MPC5 cells were differentiated into mature podocytes after incubation in RPMI-1640 medium without IFN-γ but containing 5% FBS at 37 °C for 14 days. MPC5 cells were cultured in low-glucose (5.5 mM D-glucose), HG (30 mM D-glucose), or hypertonic [5.5 mM D-glucose + 24.5 mM mannitol (MA)] medium, and treated with solvents or 25 µg/mL ADSCs-Exos combined with NC siRNA or HO-1 siRNA or FAM129B siRNA as designed for 72 h after 24 h of synchronous culture in RPMI-1640 medium with 0.2% FBS. The siRNA sequences are listed in Supplementary Table
1.
Animal treatment
C57BL/KsJ db/m (control mice, n = 10) and C57BL/KsJ db/db (spontaneous diabetes mice, n = 20) male mice were housed in a 12 h light/dark cycle under constant temperature and humidity until 12-weeks-old. After identification via periodic acid-Schiff staining, PBS or ADSCs-Exos (100 µg/mL) were injected into mice from each group through the tail vein. After 12 weeks, the mice were euthanized, and kidney tissues were removed and stored in liquid nitrogen for subsequent experiments.
Immunofluorescence
The cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. The cells were blocked with goat serum for 30 min at room temperature. Then they were incubated with the primary antibody at 4 °C overnight. This was followed by a wash with PBST, following which they were incubated at 37 °C for 1 h with a fluorescence-labeled secondary antibody (BOSTER, Wuhan, China, BA1032; 1:500). Then, the cells were stained with DAPI (Beyotime, Shanghai, China, C1002) for 5 min in the dark. Images were observed under a fluorescence microscope (Olympus, BX53) after the mounting the cells in anti-fade mounting medium (Southernbiotech, 0100-01).
Western blotting
Radioimmunoprecipitation assay buffer (Cell Signaling Technology) was mixed with phosphatase and protease inhibitors to lyse the cells, and total protein was obtained by centrifugation. Samples were quantified with a BCA kit (Beyotime, Shanghai, China, P0012S), electrophoresed on 10% SDS-PAGE gels, and transferred to a polyvinylidene fluoride membrane according to their molecular weight. After blocking with 5% milk, the membrane was incubated with primary antibodies at 4 °C. Secondary antibodies were incubated with the membranes for 1 h at room temperature. An electrochemiluminescence kit was used to visualize the chemiluminescence.
Co-immunoprecipitation (Co-IP)
After lysis with IP lysate (Beyotime, Shanghai, China; P0013) for 15 min on ice, the supernatant was collected via centrifugation. The samples were pretreated with agarose A + G and slowly shaken at 4 °C for 2 h. IgG or Keap1antibodies were added to 500 µL of total protein. The antigen-antibody mixture was slowly shaken overnight at 4 °C. Then, 30 µL of 50% agarose protein A + G was added to each tube, and the reaction was carried out for 6 h at 4 °C. After instantaneous high-speed centrifugation, the precipitate was collected and washed with precooled PBS. Then, 2× loading buffer was added to the precipitate and resuspended, followed by a metal bath reaction at 100 °C for 5 min and immediate cooling on ice. The supernatant was collected via centrifugation at 12,000 rpm for 10 min. The input group without IgG or Keap1 antibodies was used as a control. Protein samples from the input, anti-IgG, and anti-Keap1 groups were subjected to western blotting to detect Keap1 and Nrf2 levels.
Enzyme-linked immunosorbent assay (ELISA)
A double antibody sandwich was used to detect the levels of IL-1β (Beyotime, Shanghai, China; PI301), IL-6 (Beyotime, Shanghai, China; PI326), and TNF-α (Beyotime, Shanghai, China; PT512). Samples or different standard concentrations were added to a 96-well plate and incubated for 120 min at room temperature. Afterwards, each well was incubated for 60 min at room temperature with 100 µL biotinylated antibody. The plate was then incubated in the dark for 20 min at room temperature with 100 µL of horseradish peroxidase-labeled streptavidin per well. The A450 value was determined immediately after mixing the TMB solution with the termination solution at room temperature in the dark for 20 min.
Reactive oxygen species (ROS) assay
First, 2 × 105 cells were seeded in 12-well plates and cultured overnight at 37 °C in a 5% CO2 incubator. Diluted DCFH solution (Beyotime, S0033S-1) was added after the cells were washed with PBS and collected via centrifugation. The mixture was mixed every 3 min for 21 min of incubation at 37 °C, and the dyes were removed using serum-free medium after incubation. The resuspended cells were stimulated with ROS-positive control (Beyotime, S0033S-2) for 20 min. Subsequently, flow cytometry was performed.
Malondialdehyde (MDA) assay
The MDA content was detected using the TBA method (Nanjing Jiancheng Bioengineering Institute, A003-1). Following the addition of the kit extract to the sample, the working solution was added, and the supernatant was collected after 40 min in a water bath at 95 °C. Absorbance was measured at 532 nm. The standard substance detection results were used to calculate MDA concentrations.
Statistical analysis
Prism 9.0 was used to statistically analyze the data. All data are presented as means ± standard deviation (SD). One-way analysis of variance was used for statistical analysis between the groups. Differences were considered significant at P < 0.05.
Discussion
The prevalence of DN has been increasing year by year and has become one of the leading causes of end-stage renal disease (ESRD) [
28]. At present, the treatment of diabetic nephropathy is mainly through symptomatic treatment such as lowering blood sugar, controlling blood pressure and reducing proteinuria. There is still a lack of effective targeted therapeutic drugs for diabetic nephropathy [
2]. Accumulating studies have shown that MSCs play a role in promoting tissue regeneration [
29], immune regulation [
3], anti-inflammation [
30], pro-angiogenesis [
31], and anti-apoptosis [
32] through cell-cell interactions and the secretion of growth factors, cytokines, chemokines, cell adhesion molecules, lipid mediators, hormones, exosomes, microvesicles, and other regulatory molecules, such as miRNA, which have demonstrated a potential therapeutic role in a variety of diseases [
33‐
35]. ADSCs-Exo can ameliorate HG-induced podocyte injury and DN progression, mainly through the mir-486-Smad2-autophagy and the miR-215-5p-ZEB2 signal transduction pathways [
24]. Here, we showed that ADSCs-Exos could attenuate HG-induced inflammatory cytokine secretion and lipid peroxidation in vitro and in vivo.
Inflammatory factors induce podocyte injury and promote DN progression through nuclear factor kappa B, toll-like receptors, and adenosine 5’-monophosphate-activated protein kinase signaling pathways [
36]. Stem cell-derived exosomes have the potential to ameliorate the destructive effects of inflammatory factors and alleviate tissue damage, suggesting that the effect of ADSCs-Exo on podocyte injury and DN progression may be closely related to its anti-inflammatory and antioxidant functions [
37]. We have previously found that HO-1 can inhibit podocyte apoptosis by up-regulating autophagy level [
38]. It has also been reported that up-regulation of Nrf2/HO-1 signaling can induce mitochondrial autophagy, oxidative stress, inflammation, apoptosis and angiogenesis in diabetic nephropathy rats[
39‐
42]. Here, we found that HG significantly reduced HO-1 expression in MPC5 cells, which was partially reversed by ADSCs-Exo treatment. Our results showed that inhibiting HO-1 expression reversed the anti-inflammatory and anti-oxidative effects of ADSCs-Exos on podocytes, confirming that ADSCs-Exo could improve HG-induced podocyte inflammation and oxidative stress by up-regulating HO-1 expression.
As a key regulatory pathway for HO-1, Keap1/Nrf2/ARE regulates the transcription of many antioxidant genes under stress-inducing, inflammatory, and pro-apoptotic conditions [
43]. Liu et al. [
44] found that Nrf2 deficiency exacerbates diabetic kidney disease in compound mutant mice. Exosomes can exert anti-inflammatory and anti-oxidative effects by regulating the Nrf2 pathway [
45,
46]. We showed that ADSCs-Exos could reverse HG-induced Nrf2 reduction in vitro and in vivo, indicating that ADSCs-Exo can regulate high glucose-induced HO-1 dependent anti-inflammatory and anti-oxidative function in podocytes, which is related to the up-regulation of Nrf2 expression. Nrf2 binds to Keap1 under physiological conditions, resulting in increased ubiquitination on Nrf2 and promoting Nrf2 degradation through the ubiquitin-proteasome pathway [
47,
48]. Under oxidative stress, Nrf2 was transported to the nucleus and involved in the transcription of a series of antioxidant genes [
49]. We found that Nrf2 content in podocytes was not altered by HG-stimulation under MG132 treatment. However, Nrf2 expression in podocytes decreased, while Keap1 expression was increased in podocytes under HG treatment. It is speculated that HG promotes the ubiquitination of Nrf2 by increasing Keap1 expression, which mediates Nrf2 degradation through the ubiquitin-proteasome pathway, and eventually leads to the reduction of Nrf2 content in podocytes. This may be the reason why HG reduces intracellular Nrf2 expression despite activating oxidative stress in podocytes. In addition, ADSCs-Exo reduced Keap1 expression in podocytes under high glucose treatment, but this regulatory effect was abolished under MG132 treatment. These results indicated that ADSCs-Exo could reduce Keap1 content and block Keap1-mediated Nrf2 degradation, which eventually led to Nrf2 accumulation in podocytes under high glucose treatment. Interestingly, Co-IP showed that ADSCs-Exos reversed the HG-induced promotion of Keap1 and Nrf2 binding in podocytes.
FAM129B is an antioxidant protein with anti-apoptotic effects in tumors, which can compete with Nrf2 and bind Keap1 to reduce Nrf2 ubiquitination, so as to activate the Nrf2 pathway [
25,
50]. Here, FAM129B expression decreased in HG-stimulated podocytes, and FAM129B knock-down enhanced HO-1 and Nrf2 expression, inflammatory factor secretion, and ROS content in podocytes. This suggests that HG could inhibit FAM129B expression, block the binding between FAM129B and Keap1, and enhance the binding between Keap1 and Nrf2, resulting in the reduction of Nrf2 expression and the impairment of HO-1 mediated anti-inflammatory and antioxidant functions. ADSCs-Exo significantly reversed the inhibition of FAM129B expression in high glucose-stimulated podocytes. In addition, the inhibitory effect of ADSCs-Exo on the decrease of HO-1 and Nrf2 expression and the increase of inflammatory factor secretion and ROS content in HG-stimulated podocytes could be reversed by FAM129B knock-down.
However, as ADSCs-Exos are mainly carriers of active components, it is unclear what specific compounds they contained. Detailed studies are needed to clarify the specific components of ADSCs-Exos that inhibit inflammation and oxidative stress.
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