关键词: Klotho, 外泌体, 脂肪间充质干细胞, 急性肾损伤, 犬

Abstract:

The study aims to investigate the therapeutic effect of exosomes from canine adipose derived mesenchymal stem cells overexpressing Klotho (KL-Exos) on acute kidney injury (AKI) in canines. Construct a recombinant plasmid pCDH-EF1-copGFP-T2A-PURO-Klotho that overexpresses Klotho (EF1-KL recombinant plasmid), and transfect it into canine adipose derived mesenchymal stem cells (cADMSCs). Collect KL-Exos by ultracentrifugation of cell culture medium. Select 8 male Chinese field dogs around one year old with similar physical condition and randomly divide them into 4 groups: normal control group (NC), acute kidney injury group (AKI), Exos treatment group (Exo), and KL-Exos treatment group (KL-Exo). AKI group, Exo group, and KL-Exo group were injected with gentamicin sulfate at a dose of 60 mg·kg^-1, twice daily for 5 consecutive days; the Exo group and KL-Exo group were treated with 300 μg of Exos or KL-Exos injected on days 6, 9, and 12, respectively; the NC group and AKI group were injected with the same dose of physiological saline. After the treatment is completed, the treatment effect is comprehensively evaluated through imaging, serology, anatomy, histology and molecular biology testing in sequence. The sequencing results indicate the successful construction of the EF1-KL recombinant plasmid. Compared to the empty vector group, the Klotho expression level of cADMSCs transfected with EF1-KL recombinant plasmid was significantly upregulated (P < 0.001), indicating the successful establishment of cADMSCs overexpressing Klotho (KL-cADMSCs).The precipitate obtained by ultracentrifugation of the cell culture medium was detected to have a particle size of around 150 nm and a "tea tray like" appearance, which also express exosomes marker proteins CD63 and Alix, indicating that the precipitate belongs to exosomes. The treatment results showed that compared with AKI group canines, Exo group and KL-Exo group canines had significantly improved renal function, improved renal morphology, texture and tissue structure, and decreased renal oxidative stress. Compared to the Exo group canines, the serum creatinine (Cre) concentration (P < 0.000 1) and urea nitrogen (Bun) concentration (P < 0.000 1) of the KL-Exo group canines significantly decreased; B-ultrasound examination showed no obvious hydronephrosis; the kidneys are reddish brown in color and have a soft and smooth texture; the results of hematoxylin-eosin (HE) staining showed that the renal tubules were arranged neatly and no lesions such as congestion were observed; the concentration of superoxide dismutase (SOD) in the kidneys significantly increased (P < 0.000 1), while the concentrations of hydrogen peroxide (H₂O₂) (P < 0.000 1) and malondialdehyde (MDA) (P < 0.01) significantly decreased; the gene expression level of kelch-like ECH-associated protein 1 (Keap1) in the kidneys was significantly upregulated (P < 0.05), and the downward trend of nuclear factor erythroid 2-related factor 2 (Nrf2) gene expression level was more pronounced. KL-Exos mainly treat AKI in canines by reducing oxidative stress in kidneys and enhancing the body's antioxidant capacity; the Keap1-Nrf2 signaling pathway may be a key pathway for KL-Exos to treat AKI.

Key words: Klotho, exosomes, adipose mesenchymal stem cells, acute kidney injury, canines