Mesenchymal Stem Cells Attenuate Renal Fibrosis via Exosomes-Mediated Delivery of microRNA Let-7i-5p Antagomir

Abstract

Background: Renal fibrosis is a chronic and progressive process affecting kidneys in chronic kidney disease (CKD). Mesenchymal stem cells-derived exosomes (MSCs-Exo) have been shown to alleviate renal fibrosis and injury, but the mechanism of MSCs-Exo-induced renal protection remains unknown.

Methods: In this study, MSCs were transfected with let-7i-5p antagomir (anti-let-7i-5p), and then exosomes were isolated from the transfected MSCs to deliver anti-let-7i-5p oligonucleotides to inhibit the level of let-7i-5p in kidney tubular epithelial cells (NRK-52E).

Results: In both NRK-52E cells stimulated by TGF-β1 and the mouse kidneys after unilateral ureteral obstruction (UUO), we demonstrated increased level of let-7i-5p. In addition, MSCs-Exo can deliver anti-let-7i-5p to reduce the level of let-7i-5p in NRK-52E cells and increase the expression of its target gene TSC1. Moreover, exosomal anti-let-7i-5p reduced extracellular matrix (ECM) deposition and attenuated epithelial-mesenchymal transition (EMT) process in transforming growth factor beta 1 (TGF-β1)-stimulated NRK-52E cells and in the kidneys of UUO-treated mice. Meanwhile, mice received exosomal anti-let-7i-5p displayed reduced renal fibrosis and improved kidney function when challenged with UUO. Furthermore, exosomal anti-let-7i-5p promoted the activation the tuberous sclerosis complex subunit 1/mammalian target of rapamycin (TSC1/mTOR) signaling pathway in vivo and in vitro.

Conclusion: In conclusion, exosomal anti-let-7i-5p from MSCs exerts anti-fibrotic effects in TGF-β1-induced fibrogenic responses in NRK52E cells in vitro as well as in UUO-induced renal fibrosis model in vivo. These results provided a novel perspective on improving renal fibrosis by MSCs-Exo.

Keywords: chronic kidney disease; exosomes and microRNAs; mesenchymal stem cells; renal fibrosis.

Figure 1

Let-7i-5p level in in vitro and in vivo model of renal fibrosis. (A) Overlapping DEMs were identified using R language. (B) NRK52E cells were treated with 10 ng/mL TGF-β1. RT-qPCR was used to detect the level of let-7i-5p, collagen 1α1 and fibronectin in cells (n = 3). **P<0.01 compared with control group. (C) RT-qPCR was used to detect the level of let-7i-5p, collagen 1α1 and fibronectin in kidneys of UUO mice (n = 3). **P<0.01 compared with sham group. The data were statistically analyzed using Student’s t-test.

Figure 2

Isolation and characterization of exosomes. (A) TEM of exosomes derived from MSCs. (B) NTA was used to measure the size distribution of MSCs-Exo. (C) Western blotting analysis of CD9 and CD81 in MSCs and their exosomes (n = 3).

Figure 3

Anti-let-7i-5p can be transferred from MSCs to NRK52E cells via exosomes. (A) RT-qPCR was used to detect the level of let-7i-5p in MSCs transfected with let-7i-5p antagomir (n = 3). **P < 0.01, compared with NC group. (B) RT-qPCR was used to detect the level of let-7i-5p in MSCs/anti-let-7i-5p exosomes or MSCs/NC exosomes (n = 3). **P < 0.01, compared with MSC/NC group. (C) NRK52E cells were indirectly co-cultured with MSCs/anti-let-7i-5p or MSCs/NC. RT-qPCR was used to detect the level of let-7i-5p in cells (n = 3). *P < 0.05, **P < 0.01, compared with untreated NRE52E group. (D) NRK52E cells were co-cultured with MSCs transfected with cy3-tagged let-7i-5p or GW4869-treated transfected MSCs. Fluorescence microscopy was used to observe exosomal transfer of let-7i-5p to NRK52E cells. The significance between two or more groups was analyzed by Student’s t-test or one-way ANOVA respectively.

Figure 4

MSCs-secreted anti-let-7i-5p inhibited the ECM deposition and EMT process in TGF-β1-treated NRK52E cells. (A) NRK52E cells, with the addition of TGF-β1, were indirectly co-cultured with MSCs/anti-let-7i-5p or MSCs/NC. Expressions of collagen 1α1, fibronectin and α-SMA in NRK52E cells were detected with Western blotting (n = 3). The relative expressions of collagen 1α1, fibronectin and α-SMA were quantified via normalization to β-actin. **P < 0.01, compared with NRK52E group; ^#P < 0.05, ^##P < 0.01, compared with the TGF-β1 group. (B) NRK52E cells, with the addition of TGF-β1, were treated with MSCs/anti-let-7i-5p exosomes or MSCs/NC exosomes. Expressions of collagen 1α1, fibronectin and α-SMA in NRK52E cells were detected with Western blotting (n = 3). The relative expressions of collagen 1α1, fibronectin and α-SMA were quantified via normalization to β-actin. **P < 0.01, compared with untreated group; ^#P < 0.05, ^##P < 0.01, compared with the TGF-β1 group. The significance between four groups was analyzed by one-way ANOVA.

Figure 5

TSC1 is a functional target of let-7i-5p in NRK52E cells. (A) Schematic diagram of binding sites between let-7i-5p and TSC1, as well as the mutation of binding sites in TSC1. (B) Luciferase assay of cells transfected with TSC1-wild type (WT) or TSC1-mutant (MT) reporter together with let-7i-5p or NC. **P < 0.01, compared with vector-control group. (C) NRK52E cells were treated with MSCs/anti-let-7i-5p exosomes or MSCs/NC exosomes. RT-qPCR was used to detect the level of TSC1 in cells (n = 3). **P < 0.01, compared with MSCs/NC-Exo group. (D and E) NRK52E cells, with the addition of TGF-β1, were treated with MSCs/anti-let-7i-5p exosomes or MSCs/NC exosomes. Expressions of TSC1, p-mTORC1, mTORC1, p-p70S6K, p70S6K, p-4E-BP1 and 4E-BP1 in NRK52E cells were detected with Western blotting (n = 3). The relative expressions of TSC1, p-mTORC1, p-p70S6K and p-4E-BP1 were quantified via normalization to β-actin, mTORC1, p70S6K and 4E-BP1. **P < 0.01, compared with untreated group; ^#P < 0.05, ^##P < 0.01, compared with the TGF-β1 group. The significance between three or more groups was analyzed by one-way ANOVA.

Figure 6

In vivo distribution of MSCs/Exo. In vivo fluorescent images of mice with sham-operated controls or UUO injury after intravenous injection of PKH67 labeled MSCs-Exo at different time point.

Figure 7

MSCs/anti-let-7i-5p exosomes alleviated renal fibrosis in UUO mice. (A) ELISA assay was used to measure the levels of BUN and CR in serum of mice and the level of CR in urine of mice. (B) Measurement of eGFR in mice. (C) H&E and Masson’s trichrome staining assay was used to analyze renal fibrosis and collagen deposition (magnification, ×200). Red arrows, glomerular injury; black arrows, renal interstitial edema; blue arrow, collagen fibers. (D) Total renal fibrotic area was measured by Image-Pro Plus. **P < 0.01, compared with Sham group; ^#P < 0.05, ^##P < 0.01, compared with the UUO group. The significance between four groups was analyzed by one-way ANOVA.

Figure 8

MSCs/anti-let-7i-5p exosomes decreased the expressions of EMT-related proteins in UUO mice. (A and B) IHC staining of collagen 1α1, fibronectin and α-SMA (indicated by arrowheads) in the kidneys of mice (magnification, ×200; n = 3). **P < 0.01, compared with Sham group; ^#P < 0.05, ^##P < 0.01, compared with the UUO group. The significance between four groups was analyzed by one-way ANOVA.

Figure 9

MSCs/anti-let-7i-5p exosomes alleviated renal fibrosis in UUO mice via upregulation of TSC1. (A) Expressions of TSC1, p-mTORC1, mTORC1, p-p70S6K, p70S6K, p-4E-BP1 and 4E-BP1 in the kidneys of mice were detected with Western blotting (n = 3). (B) The relative expressions of TSC1, p-mTORC1, p-p70S6K and p-4E-BP1 were quantified via normalization to β-actin, mTORC1, p70S6K and 4E-BP1. (C) RT-qPCR was used to analyze the level of let-7i-5p in the kidneys of mice (n = 3). **P < 0.01, compared with Sham group; ^#P < 0.05, ^##P < 0.01, compared with the UUO group. The significance between four groups was analyzed by one-way ANOVA.