Melatonin improves therapeutic potential of mesenchymal stem cells-derived exosomes against renal ischemia-reperfusion injury in rats

Abstract

Renal ischemia-reperfusion injury (RIRI) is one of the main causes for acute kidney injury (AKI). Many previous attempts failed to adopt a suitable treatment regimen for AKI. Recently, combined melatonin (Mel) and mesenchymal stem cell (MSC)-derived exosomes (Exo) therapy gave a promising therapeutic option for acute liver ischemic injury, however this treatment approach has not been tested against RIRI yet. This study tested the hypothesis that administration of exosomes derived from MSCs preconditioned with Mel gave best protection against RIRI as compared to therapy by MSCs or exosomes derived from non-preconditioned MSCs. Female adult rats (n = 60) equally divided into control group, sham group, RIRI group (induced by bilateral renal arteries clamping), RIRI + MSCs group (1 × 10⁶ bone marrow derived MSCs), RIRI + Exo group (250 μg Exo derived from no-preconditioned MSCs), and RIRI + Mel + Exo group (250 μg Exo derived from Mel preconditioned MSCs). MSCs or Exo was bilaterally injected once in each renal artery during reperfusion. The obtained results revealed notable improvement in RIRI following all treatment (MSCs, Exo, and Exo + Mel) with best improvement in Exo + Mel group as evidenced by: 1) decreased kidney injury histopathological score; 2) reduced blood levels of kidney damage markers [blood urea nitrogen (BUN) and creatinine]; 3) declined oxidative stress status (MDA level, HIF1α gene, and NOX2 protein); 4) increased anti-oxidant status (HO1 gene, and SOD, CAT, GPX activities); 5) declined apoptosis (caspase 3 activity and mRNA, and PARP1, Bax genes), 6) induced anti-apoptotic effect (Bcl2 gene); 7) inhibition of inflammation (decreased MPO activity and ICAM1, IL1B, NFkB genes and increased IL10 genes); 8) improved regeneration (bFGF, HGF and SOX9 proteins); and 9) enhanced angiogenesis (VEGF gene). These data indicate that treatment with exosomes derived from MSCs preconditioned with melatonin gave best protective effect against renal ischemia-reperfusion injury as compared to therapy by non-preconditioned MSCs or their exosomes.

Keywords: Melatonin; exosomes; mesenchymal stem cells; renal ischemia.

Figure 1

Characterization and identification of cultured MSCs and their exosomes. A. MSCs with their characteristic fusiform (fibroblast-like) shape were grown after passage 8, Scale bar = 50 μm. B. Characterization of MSCs by flow cytometer analysis showed that 92.26% and 97.05% of the bone marrow derived MSCs were positive to CD44 and CD90, respectively, while only 1.93% positive for CD34 which considered as negative results. C. Transmission electron microscopic examination shows small nanovesicles (20-130 nm) in the sample isolated from the MSCs culture media by ultracentrifugation. D. Western blot analysis of the exosomal protein shows presence of CD63 and CD81.

Figure 2

H&E staining and the analysis of renal damage score. (A, B) Control group; (C, D) Sham group; (E, F) RIRI group; (G, H) MSCs group; (I, J) Exo group; and (K, L) Mel + Exo group at 3 days (A, C, E, G, I, K) and 4 weeks (B, D, F, H, J, L) post-ischemia. In all images, black arrows and arrowheads refer to renal tubules and glomeruli, respectively. (M and N) Pathological scoring of kidney at 3 days (M) and 2 weeks (N). Values were means ± SEM (n = 10/group and 5/each time point). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars = 50 µm in (E and F); 100 µm in all remaining images.

Figure 3

Effect of MSCs or exosomes treatment on oxidative stress (MDA, HIF1α gene, NOX2 protein) and antioxidant (SOD, CAT, GPX enzymes and HO1 gene) status of kidney in RIRI rats at 3 days and 4 weeks time points. Values are expressed as mean ± SEM. Columns carrying different letters are significantly different at P < 0.05.

Figure 4

Effect of MSCs or exosomes treatment on apoptosis (caspase 3 activity and mRNA level, Bax, PARP1, and Bcl2 genes). Data presented as fold change (mean) ± SEM from the control group. Columns carrying different letters are significantly different at P < 0.05.

Figure 5

Effect of MSCs or exosomes treatment on inflammation (MPO, and mRNA levels of ICAM-1, IL1β, NFκB, and IL10). Data presented as fold change (mean) ± SEM from the control group. Columns carrying different letters are significantly different at P < 0.05.

Figure 6

Effect of MSCs or exosomes treatment on regeneration (bFGF, HGF, and SOX9 proteins) and angiogenesis (VEGF gene). Data presented as fold change (mean) ± SEM from the control group. Columns carrying different letters are significantly different at P < 0.05.