Exosomes Derived from BM-MSCs Mitigate the Development of Chronic Kidney Damage Post-Menopause via Interfering with Fibrosis and Apoptosis

Abstract

The rate of chronic kidney disease (CKD) is increasing globally, and it is caused by continuous damage to kidney tissue. With time the renal damage becomes irreversible, leading to CKD development. In females, post-menopause lack of estrogen supply has been described as a risk factor for CKD development, and studies targeting post-menopause CKD are scarce. In the present study, we used exosomes isolated from bone marrow mesenchymal stem/stromal cells (BM-MSCs) to test their therapeutic potential against the development of CKD. At first, the menopause model was achieved by surgical bilateral ovariectomy in female albino rats. After that, 100 µg of exosomes was given to ovariectomized rats, and the study continued for 2 months. Changes in urine volume, urine protein content, kidney function biochemical parameters (creatinine and BUN), kidney antioxidant parameters (SOD, GPx and CAT), histological changes, immunohistochemical levels of caspase 3, and the gene expression of NGAL (related to kidney damage), TGFβ1 and αSMA (related to fibrosis and EMT), and caspase 3 (related to apoptosis) were studied. After the ovariectomy, the occurrence of CKD was confirmed in the rats by the drastic reduction of serum estrogen and progesterone levels, reduced urine output, increased urinary protein excretion, elevated serum creatinine and BUN, reduced GPx SOD, and CAT in kidney tissue, degenerative and fibrotic lesions in the histopathological examination, higher immunohistochemical expression of caspase 3 and increased expression of all studied genes. After exosomes administration, the entire chronic inflammatory picture in the kidney was corrected, and a near-normal kidney structure and function were attained. This study shows for the first time that BM-MSCs exosomes are potent for reducing apoptosis and fibrosis levels and, thus, can reduce the chronic damage of the kidneys in females that are in their menopause period. Therefore, MSCs-derived exosomes should be considered a valuable therapy for preserving postmenopausal kidney structure and function and, subsequently, could improve the quality of females' life during menopause.

Keywords: caspase 3; chronic renal injury; exosome therapy; fibrosis; nephroprotective; renoprotective.

Figure 1

Characterization of isolated BM-MSCs. (A) Isolated BM-MSCs in culture at passage 3 with the typical fibroblast-like shape. (B) Flow cytometric evaluation of BM-MSCs markers seen as a positive expression of CD90 and CD105, and negative expression of endothelial CD34 and hematopoietic CD45 markers.

Figure 2

Characterization of exosomes obtained from BM-MSCs. (A) TEM examination showing the presence of exosomes and their diameter is less than 100 nm. (B) Western blot analysis of exosomes’ markers CD63 and CD81. (C) Scheme outlining the experimental setup of the study.

Figure 3

Changes in serum estrogen and progesterone levels in study rats. CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. Different letters denote statistical significance (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 4

Serum biochemical parameters (creatinine and blood urea nitrogen) following treatment with MSCs-derived exosomes in study groups. The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. Columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 5

Antioxidant parameters in kidney tissue, glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) in the different study groups. The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. Columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 6

Changes in glomerular filtration rate (GFR) and urinary protein excretion in the different study groups. The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in Their tail vein. Columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 7

Representative micrographs for the histological evaluation of the lesions in the kidneys of different studied groups. (A) Control group, (B) Sham group, (C) CKD group, and (D) CKD + Exosomes group; The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. Black arrow head = normal glomeruli, Yellow arrow head = shrunken glomeruli with degenerated glomerular cells, wide intraglomerular space and edema in some glomeruli, Black arrow = normal renal tubules, Red arrow = degenerated renal tubular epithelial cells, Orange arrow = fibrosis, and Curved arrow = mononuclear cells infiltration. The right bar graph shows the percentage of damaged glomeruli (shrunken with/without edema) and the percentage of focal fibrosis in the different study groups, columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 8

Representative micrographs showing the expression of caspase 3 in kidney tissue by immunohistochemistry in the different studied groups. The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. The right bar graph shows the intensity of caspase 3 staining in the different groups, columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).

Figure 9

Changes in the mRNA expression of (A) neutrophil gelatinase-associated lipocalin (NGAL), (B) transforming growth factor beta 1 (TGFβ1), (C) caspase 3 and (D) smooth muscle alpha-actin (αSMA) genes in the different studied groups. The CKD + Exosomes group received an intravenous injection of 100 µg protein-equivalent of exosomes in their tail vein. Columns with different letters denote significant differences between groups (p < 0.05, using Tukey’s HSD post-hoc test).