Pan PPAR agonist stimulation of induced MSCs produces extracellular vesicles with enhanced renoprotective effect for acute kidney injury

Abstract

Background: Acute kidney injury (AKI) has a complex pathophysiology and imposes serious health concerns worldwide. Extracellular vesicles (EVs) derived from induced mesenchymal stem cells (iMSCs) have been recognized as novel cell-free therapeutics for various inflammatory and degenerative disorders. In this study, we investigated whether iMSCs stimulated with a pan-peroxisome proliferator-activated receptor (PPAR) agonist could enhance the therapeutic efficacy of EVs against AKI.

Methods: Human iMSCs were primed with or without lanifibranor, a PPAR agonist for 24 h, and EVs were collected after an additional 24 h. The basic characteristics of EVs were evaluated using cryo-transmission electron microscopy imaging, immunoblot detection of EV markers, nanoparticle tracking analysis, and localization in AKI kidneys. In vitro, the potential of the EVs to promote the growth and survival of HK-2 cells undergoing cisplatin-induced apoptosis and anti-inflammatory effects in M1-polarized THP-1 was compared. Subsequently, AKI was induced in BALB/c mice using cisplatin. After 8 and 24 h of cisplatin treatment, iMSC-EVs or pan-PPAR-iMSC-EVs were injected intravascularly. At 96 h after cisplatin administration, the renoprotective effects of iMSC-EVs or pan-PPAR-iMSC-EVs in inhibiting inflammation and apoptosis were compared using serum biochemistry, histology, immunohistochemistry, and gene expression analysis by qPCR.

Results: Both EV types expressed EV markers and had typical EV morphology, and their localization in the renal tissue was confirmed. The proliferation and survival of HK-2 cells were higher in pan-PPAR-iMSC-EVs than those in iMSC-EVs. In M1-polarized THP-1 cells, the reduction in the mRNA expression of inflammatory cytokines was more significant in pan-PPAR-iMSC-EVs than that in iMSC-EVs. In the mouse model of cisplatin-induced AKI, pan-PPAR-iMSC-EVs markedly enhanced renoprotective effects compared to iMSC-EVs. Specifically, pan-PPAR-iMSC-EVs reduced tissue inflammation, immune cell infiltration, and apoptosis. Pan-PPAR-iMSC-EVs also increased renal capillary density.

Conclusion: Priming iMSCs with a PPAR agonist significantly improved the therapeutic potential of EVs by reducing inflammation and apoptosis. The reported strategy may contribute to the development of a novel cell-free option for AKI treatment.

Trial registration: Not applicable.

Keywords: Acute kidney injury; Extracellular vesicle; Induced mesenchymal stem cells; Priming.

Fig. 1

Characterization of iMSC-EVs and pan PPAR-iMSC-EVs. A Immunoblot detection of markers of extracellular vesicles in iMSC-EVs and pan PPAR-iMSC-EVs. The expression of markers for EVs (CD9, CD81, and TSG101) or organelles (calnexin) analyzed in iMSC-EVs and pan PPAR-iMSC-EVs. Full-length blots are presented in Additional file 2: Fig. S1. B The morphology of iMSC-EVs and pan PPAR-iMSC-EVs. Cryo-TEM was used for imaging. Scale bar: 100 nm. C Size distribution of iMSC-EVs and pan PPAR-iMSC-EVs measured using a nanotracking particle analyzer. D Detection of iMSC-EVs and pan PPAR-iMSC-EVs in the AKI kidney. iMSC-EVs or pan PPAR-iMSC-EVs were stained with CMTMR and subsequently intravascularly administered into mice 8 h after cisplatin treatment. After 72 h of cisplatin administration, the kidneys were harvested, and the presence of iMSC-EVs or pan PPAR-iMSCs was detected under laser confocal microscopy. E The effect of iMSC-EVs or pan PPAR-iMSC-EVs on the growth of HK2 cells. HK2 cells were cultured with iMSC-EVs or pan PPAR-iMSC-EVs under serum-free conditions for 24 (left) and 48 (right) h. The relative number of viable cells was determined by measuring the optical density (OD₄₅₀) using a CCK-8 assay. F The effect of iMSC-EVs or pan PPAR-iMSC-EVs on the survival of HK2 cells undergoing cisplatin-mediated cell death. HK2 were treated with 15 μM of cisplatin for 24 h in the presence of iMSC-EVs or pan PPAR-iMSC-EVs. The relative number of viable cells was determined by measuring the optical density (OD₄₅₀) using the CCK-8 assay

Fig. 2

Assessment of therapeutic efficacy of EVs and pan PPAR-iMSC-EVs. iMSC-EVs or pan PPAR-iMSC-EVs were administered intravenously 8 and 24 h after cisplatin treatment. After 96 h of cisplatin treatment, mice were sacrificed. A The concentration of blood urea nitrogen, serum creatinine, and body weight. B Light microscopy images of kidney sections stained with hematoxylin and eosin. Magnification: 200x. C Assessment of renal injury (N = 4); Data are presented as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. D Immunohistochemical staining of proliferating cells with anti-PCNA antibody. Magnification: 400x. Scale bar: 50 μm. *p < 0.05; ***p < 0.001

Fig. 3

Alleviation of inflammation by pan PPAR-iMSC-EVs in AKI kidney. Immunohistochemical staining of NGAL (A) and TNF-α (B). (C) The protein expression of NGAL, TNF-α, phosphorylated P38 and ERK1/2 was compared among AKI animals that received the vehicle, iMSC-EV, or pan PPAR-iMSC-EVs. Each lane represents a sample from one animal. After antibodies corresponding to each protein were used for immunoblotting, the relative expression of the proteins was normalized against that of β-actin and quantified using ImageJ software; N = 4; Data are presented as mean ± sd. *p < 0.05; ***p < 0.001; ****p < 0.0001. N represents normal animals that had not undergone cisplatin-mediated AKI. Full-length blots are presented in Additional file 2: Figs. S2, S3

Fig. 4

Immunohistochemical detection of inflammatory cells and measurement of capillary density in the AKI mice. A The expression of immune cell markers (CD45, F4/80, and Ly6G) and blood vessels (CD31) are shown; Scale bar: 100 μm. For CD31 staining, enlarged images of each figure are also provided. B Comparison of the number of infiltrated immune cells and capillary structures in AKI kidneys. Data are presented as mean ± sd; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Effect of pan PPAR-iMSC-EVs on apoptotic injury in AKI. A Immunohistochemical detection of BAX protein expression in the kidney from AKI mice. Scale bars are 100 μm. B Detection of cleaved caspase-3-expressing cells in the kidney of AKI mice that received pan PPAR-iMSC-EVs; N = 4. Scale bar: 100 μm. Data are presented as mean ± sd. *p < 0.05; ****p < 0.0001. C Immunoblot analysis of apoptosis markers in AKI kidney. Single lane represents lysate from a single animal. Markers of ER stress (CHOP), apoptosis (BAX, caspase caspase 3) and necroptosis (RIP3, MLKL) were detected using corresponding antibodies. Their relative expression was normalized against that of β-actin and then quantified using ImageJ software; N = 4. Data are presented as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Full-length blots are presented in Additional file 2: Fig. S5