Epsin1-mediated exosomal sorting of Dll4 modulates the tubular-macrophage crosstalk in diabetic nephropathy

Abstract

Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.

Keywords: Dll4; Epsin1; diabetic nephropathy; exosome; inflammation.

Graphical abstract

Figure 1

Identification of enhanced Dll4 in urinary exosomes from DN patients (A) Representative transmission electron microscope (TEM) image of urinary exosomes released by DN patients. Scale bar, 100 nm, 200 nm. (B) Size distribution and number of DN urinary exosomes presented with nanoparticle tracking analysis (NTA). (C) The volcano plot of proteins analyzed from urinary exosomes. The volcano plot was constructed with log2 expression of fold change and the corresponding tempered log2 p value of all proteins. Red dots represent upregulated proteins and blue dots represent downregulated proteins. (D and E) Biological process and KEGG pathway of differentially expressed proteins identified by proteome. (F) A heatmap based on top 15 differentially expressed proteins according to their significance among signal transduction mechanism. Red represents strong enrichment and green represents weak enrichment. (G–G2) Immunoblots using urinary exosome proteins (5 μg each) from normal controls(NCs) and DN patients against exosome markers (CD9, CD81, Alix) and Dll4, followed by their densitometry analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.

Figure 2

Levels of Epsin1, Dll4, and N1ICD were significantly enhanced in the urinary exosomes and kidney tissue of DN patients and were positively correlated with tubulointerstitial damage (A–B2) Immunoblot against Epsin1, Dll4, N1ICD, and the exosome marker Alix using urinary exosome preparations (U-exo), followed by their densitometric and linear correlation analysis. (C) Histologic (hematoxylin-eosin [HE] and periodic acid-Schiff [PAS] staining) and immunohistologic (CD68, Epsin1, Dll4, N1ICD immunostaining) changes. Scale bar, 50 μm. (D1) Quantitative analysis of interstitial fibrosis and tubular atrophy (IFTA) scores. (E1–E4) Relative intensity of CD68, Epsin1, Dll4, and N1ICD in the kidney tissues. (F1–F3) The linear correlations analysis of Epsin1, Dll4, and N1ICD expression levels with IFTA scores. (G and H) Immunofluorescence staining of Dll4/N1ICD (green) and the macrophage marker CD68 (red) in frozen kidney sections. Scale bar, 50 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.

Figure 3

Expression of Epsin1 was positively associated with the level of Dll4/N1ICD and tubulointerstitial damage in the kidneys of db/db mice (A) Histologic (HE and PAS staining) and immunohistologic (F4/80, Epsin1, Dll4, Notch1, N1ICD immunostaining) changes in mice. Scale bar, 50 μm. (B1) Quantitative analysis of IFTA scores. (C1–C5) Relative intensity of F4/80, Epsin1, Dll4, Notch1, N1ICD in the kidney tissues. (D1–D4) The linear correlations analysis of Epsin1, Dll4, Notch1, and N1ICD expression levels with IFTA scores. (E–E3) Western blot analysis of Epsin1, Dll4, N1ICD expression of kidney with animal model, followed by their densitometric analysis. (F) Immunofluorescence staining of Dll4 (red), Epsin1 (red), and the tubule marker LTL (green) in frozen kidney sections. Scale bar, 50 μm. (G) Immunostaining of Dll4 (green), N1ICD (green), and the macrophage marker F4/80 (red) in frozen kidney sections. Scale bar, 50 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.

Figure 4

Exosomes from HG-treated HK-2 cells with Epsin1 knockdown ameliorated macrophage activation and tubulointerstitial damage in vivo (A) Typical transmission electron microscope (TEM) image of the exosomes secreted by HK-2 cells (HK-2 exo). Scale bar, 100 nm, 200 nm. (B) Size distribution and number of exosomes isolated from HK-2 cells culture medium were examined with nanoparticle tracking analysis (NTA). (C) Representative western blotting of exosome markers (including calnexin, Alix, CD9, and CD81) in HK-2-derived exosomes with normal-glucose (NG) or high-glucose (HG) treatment (also defined as NG/Ctrl-exo and HG-exo). (D) Western blotting showing that HK-2 sublines were transfected with Epsin1-knockdown (KD) lentivirus and generated Epsin1-KD exosomes upon HG treatment (called HG + Ep1 KD-exo). Scale bar, 10 μm. (E) Representative images of DiO-labeled kidney section showing the preparation of tubulointerstitial exo-injection (50 μg) mice model. Scale bar, 50 μm. (F) Representative images of PAS-stained kidney sections at day 1(D1) and day 3(D3) after Ctrl-exo or HG-exo-injection at two doses (10 μg, 50 μg), respectively. Scale bar, 50 μm. (G) Blood urea nitrogen (BUN) and serum levels of creatinine (Scr) in mice at day 3 after 50 μg exosomes administration. (H) Histological changes (PAS staining) and representative images of F4/80, iNOS, TNF-α, Dll4, and N1ICD-stained kidney sections at day 3 after 50 μg exosome administration (Ctrl-exo, HG-exo, HG + Ep1 KD-exo), respectively. Scale bar, 50 μm. (I) Immunofluorescence staining of Dll4 (green), N1ICD (green), and the macrophage marker F4/80 (red) in frozen kidney sections. Scale bar, 50 μm. (J and J1) Western blot analysis of iNOS, IL-6, N1ICD expression of kidney with exo-injected animal model, followed by their densitometric analysis. (K) Real-time quantitative PCR measuring N1ICD target gene (HES1) and M1 markers (iNOS and IL-6) in exo-injected kidney sections. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments at least and are expressed as mean ± SD.

Figure 5

Dll4-enriched exosomes originating from HG-treated HK-2 cells promoted M1 macrophage activation (A) Immunoblots against Dll4 and the exosome markers (Alix, CD9, and CD81). (B) Chemotaxis test for THP-1 macrophages when THP-1 cells were seeded in the top compartment of Transwell, separated by a porous membrane from HK-2 cells culture medium. Scale bar, 10 μm. (C) Internalization of HK-2-derived exosomes by macrophages. Exosomes were purified from the DiO-labeled HK-2 cells and were then applied to recipient THP-1 macrophages which were pretreated with 100 ng/mL PMA. Scale bar, 10 μm. (C1) Relative fluorescence intensity was calculated. (D–D2) Western blotting and quantification of iNOS, IL-6 in recipient THP-1 macrophages treated with NG-exo, HG-exo and LPS, LPS was used as a positive control. (E) Expression analysis of CD86+ and CD206+ by flow cytometry for THP-1 macrophages stimulated with NG-exo, HG-exo, and LPS. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.

Figure 6

Epsin1 modulated exosomal Dll4 derived from HG-stimulated HK-2 cells (A and A1) Western blotting for expression of Epsin1, Dll4 in HK-2 cells and their densitometric analysis. (B–C1) Immunoblotting against Alix-standardized Dll4 in exosomes secreted from Epsin1 siRNA-transfected and Epsin1-KD lentivirus-transfected HK-2 cells respectively, followed by their densitometric analysis. (D and D1) Western blotting proving HK-2 sublines were transfected with Dll4-OE lentivirus. (E and E1) Western blot analysis of exosomal Dll4 from the Dll4-OE sublines. (F and F1) Western blotting proving HK-2 sublines were transfected with both Epsin1-KD and Dll4-OE lentivirus. (G and G1) Western blot analysis of exosomal Dll4 from HK-2 subline transfected with both Epsin1-KD and Dll4-OE lentivirus. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.

Figure 7

Epsin1 knockdown in HK-2 cells alleviated macrophage N1ICD activation and M1 polarization by inhibiting HK-2-derived exosomal Dll4 (A) Transwell system where exosomes from HK-2 cells might pass through the basolateral membrane to the medium of THP-1 cells. (B–B3) Western blot analysis for expression of iNOS, Dll4, and N1ICD in THP-1 macrophages after coculturing THP-1 with HK-2 cells by Transwell system where HK-2 cells seeded upper and THP-1 cells seeded lower. (C) THP-1 cell incubation with exosomes derived from HK-2 cells. (D–D3) Immunoblot analysis for expression of iNOS, Dll4, and N1ICD in THP-1 macrophages accepting exosomes (20 μg per group) from HK-2 cell lines, respectively. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of three independent experiments and are expressed as mean ± SD.