Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Abstract

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.

Keywords: exosome; microRNA; renal fibrosis; unilateral ischemia-reperfusion.

Figure 1

Increased renal injury and fibrosis are associated with prolonged ischemic time in UIRI. (A) HE, TUNEL staining and flow cytometry of kidney tissues from the sham and UIRI groups at 14 days, 28 days (n=6). (B) Area of collagen in different groups as indicated by Masson staining. (C) Evaluation of tubular injury scores by HE staining in the sham and UIRI groups at 14 days, 28 days. (D) Evaluation of positive cell apoptotic rate by TUNEL staining. (E) Evaluation of apoptotic rate by flow cytometry. (F) Quantitative determination of collagen area in different groups. ^*P < 0.05 versus sham controls; ^#P < 0.05 versus ischemia 30 minutes.

Figure 2

Increased secretion of exosomes in the kidney following UIRI. (A) Immunofluorescence staining for CD63 and TSG-101 (red). (B) Integrated optical density (IOD) of TSG-101. (C) Integrated optical density (IOD) of CD63. (D) Double immunofluorescence staining for CD63 (red) and AQP-1 (green), respectively. (E) Western blotting of CD63 in kidney tissue and exosome at different time points after UIRI (n=6). (F) Quantitative of CD63 in kidney. (G) Quantitative of CD63 in exosome. (H) TEM image of the exosomes in the kidney tissue after UIRI. ^*P < 0.05 versus sham.

Figure 3

Inhibition of exosome secretion by Rab27a knockout and inhibitor GW4869 prevents renal fibrosis in vivo. (A, B) Rab27a knockout as confirmed by PCR screening (Targeted allele: 476 bp) and sequencing confirmation. (C) Western blot analysis of CD63 in kidney after Rab27a knockout. (D) Quantitative data of CD63 are presented. (E) Western blotting of fibronectin in kidney. (F) Quantitative data of fibronectin are presented. (G) Western blotting of α-SMA in kidney. (H) Quantitative data of α-SMA are presented. (I) Area of collagen in different groups as indicated by Masson staining. (J) Quantitative of kidney fibrotic area in different groups. (K) Western blotting of CD63 in kidney after GW4869 treatment (n=6). (L) Quantitative data of CD63 are presented. (M) Western blotting of fibronectin in kidney. (N) Quantitative data of fibronectin are presented. (O) Western blotting of α-SMA in kidney. (P) Quantitative data of α-SMA are presented. ^*P < 0.05 versus sham; ^#P < 0.05 versus UIRI.

Figure 4

Increased tubular cell-derived exosomes after hypoxia treatment mediate fibroblast activation. (A) Diagram shows the exosomes were isolated from conditioned media by ultra-centrifugation. (B) Experimental design shows exosomes were extracted from NRK-52E cells treated without (Con-Exo) or with hypoxia (Hy-Exo) incubated with NRK-49F cells (n=3). (C) TEM image of the exosomes isolated from hypoxic NRK-52E cells. (D) NTA of exosomes from hypoxic NRK-52E cells. (E) Western blotting of CD63 and TSG-101 as exosome marker. (F) Fluorescent staining image of NRK-52E cell-derived exosome being taken up by NRK-49F cells. (G) Western blotting of α-SMA after Hy-Exo treatment. (H) Quantitative of α-SMA are presented. (I) Western blotting of fibronectin after Hy-Exo treatment. (J) Quantitative of fibronectin are presented. (K) The slides of cell were subjected to immunostaining for fibronectin. Arrows indicate positive staining. (L) Representative micrographs show fibronectin expression in different groups as indicated. (M) The slides of cell were subjected to immunostaining for α-SMA. Arrows indicate positive staining. (N) Representative micrographs show α-SMA expression in different groups as indicated. ^*P < 0.05 versus Con-Exo.

Figure 5

Inhibition of exosome secretion by Rab27a knockdown and inhibitor GW4869 attenuates fibroblast activation in vitro. (A) Experimental design shows exosome from hypoxia NRK-52E cells with GW4869 or siRNA Rab27a treatment to stimulate NRK-49F cells. (B) PCR detection of Rab27a after transfection with siRNA Rab27a. (C) Western blotting of CD63 in exosome after Rab27a knockdown. (D) Quantitative of CD63 with siRNA Rab27a treatment are presented. (E) Western blotting of CD63 in exosome after GW4869 treatment. (F) Quantitative of CD63 with GW4869 treatment are presented. n=3, ^*P < 0.05 versus Con; ^#P < 0.05 versus Hy. (G) The slides of cell were subjected to immunostaining for α-SMA and fibronectin. Arrows indicate positive staining. (H) Representative micrographs show fibronectin expression in different groups as indicated. (I) Representative micrographs show α-SMA expression in different groups as indicated. (J) Fold changes in the expression levels of α-SMA and fibronectin with siRNA Rab27a treatment. (K) Fold changes in the expression levels of α-SMA and fibronectin with GW4869 treatment. n=3, ^*P < 0.05 versus Con-Exo; ^#P < 0.05 versus Hy-Exo.

Figure 6

Tubule cell-derived exosomal miR-150-5p mediate fibroblast activation by SOCS1 in vitro. (A) Exosomal miR assay analysis of NRK-52E cells after hypoxia treatment. (B) Scatter plot showed exosomal miR changes following hypoxia treatment. (C) PCR detection of miR-150-5p in exosomes and NRK-52E cells after hypoxia treatment. (D) PCR detection of miR-150-5p in NRK-52E cells after Anti-miR-150 LNA treatment. (E) PCR detection of α-SMA and fibronectin. (F) Potential miR-150 binding site in 3'-UTR of SOCS1 mRNA. (G) Luciferase activity in NRK-49F cells transfected with negative control or miR-150-5p mimic together with reporter vector containing SOCS1-mut binding sequences. (H) Immunoblot analysis of SOCS1 expression. (I) Densitometry analysis of SOCS1 expression. n=3, ^*P < 0.05 versus Con-Exo; ^#P < 0.05 versus Hy-Exo.

Figure 7

Tubular cell-derived exosomes promote renal fibrosis by inhibiting SOCS1 after UIRI in vivo. (A) Diagram shows NRK-52E derived exosomes were injected i.v. at days 1,3,5 and 7 after UIRI. (B) PKH26-labeled exosomes in kidney with UIRI kidney as shown by an in vivo imaging system. (C) Paraffin sections were subjected to fibronectin and α-SMA immunostaining, Sirius Red Staining (n=6). Arrows indicate positive staining. (D) Representative micrographs showed collagen deposited for α-SMA and fibronectin immunostaining and Sirius Red Staining in different groups as indicated. (E) Expression of SOCS1 were examined by Western blot analysis after exosome injection. (F) Densitometry analysis of SOCS1 expression. ^*P < 0.05 versus sham; ^#P < 0.05 versus Con-Exo; ^ΔP < 0.05 versus Hy-Exo.

Figure 8

Summary of the role of exosome in renal fibrosis after UIRI.