The Role Of Epsin1-Mediated Exosomal Dll4 In Tubular-Macrophage Crosstalk Linked With Diabetic Nephropathy

Diabetic nephropathy(DN)is the leading cause of end-stage renal disease(ESRD)worldwide.Increasing evidence has shown that renal tubular epithelial cells(TECs)can activate macrophages through the secretion of exosomes.Dll4 can serve as a key mediator in exosomes and be transferred between different types of cells.Epsin1 is related to diabetes,and as a vital endocytic protein,it is essential for the canonical Dll4/Notch1 signaling transduction.However,the role of Epsin1 in exosome-dominated TEC-to-macrophage communication and the tubulointerstitial inflammation under DN conditions remains unclear.Objective:This study is aimed to identify the differentially expressed proteins in urinary exosome from type 2 DN patients by mass spectrometry and subsequently focuses on the correlation analysis for exosomal Dll4 and macrophage infiltration and activation and tubulointerstitial inflammation.Importantly,another goal of this research is to explore the mechanism that Epsin1-mediated sorting of Dll4 into TEC-exosomes modulates the phenotype shift of renal macrophages under hyperglycemic conditions.Methods:1.Ultracentrifugation was used to isolate and purify exosomes.The extracted exosomes were further identified by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and western blotting(WB).The differentially expressed proteins in urinary exosome from DN patients were analyzed by mass spectrometry and confirmed by western blotting.2.Immunohistochemistry and immunofluorescence staining were utilized to observe the macrophages infiltration into renal tissue of type 2DN patients and db/db mice and the expression of Epsin1,Dll4 and N1 ICD in the regions of tubules and interstitium.Combining with western blotting,the expression of Epsin1,Dll4 and N1 ICD was measured.In addition,correlation analysis was conducted between Epsin1 and Dll4,N1 ICD,interstitial fibrosis and tubular atrophy(IFTA).3.By transfecting Epsin1 knockdown(KD)lentivirus with HK-2cells,Epsin1 KD-HK-2 cell line was constructed and cultured with high glucose(30m M,24h)to extract their exosomes.Afterwards,those exosomes were injected intrarenal into C57BL/6 mice in order to evaluate the effects of Epsin1 KD-HK-2-derived exosomes on renal function,inflammation,macrophage activation and renal Notch1 signaling transduction in vivo.4.Three HK-2 cell lines were constructed using Epsin1 knockdown(KD)and Dll4 overexpressed(OE)lentiviruses: Epsin1 KD-,Dll4 OE-,Epsin1 KD+Dll4 OE-HK-2 cell line.Methods of the direct incubation of HK-2 exosomes with THP-1 macrophages or Transwell system where HK-2 cells and THP-1 macrophages cocultured were used to induce phenotypic differentiation of macrophages in vitro,which was further detected by flow cytometry and western blotting.Results:1.1644 differentially expressed proteins were detected in urinary exosomes from DN patients,among which Dll4 was significantly increased and was positively correlated with Epsin1 and N1 ICD as DN progressed.2.Compared with the control group,the expressions of macrophage marker(CD68 or F4/80)in renal tissues and Dll4/N1 ICD in both renal tubules and interstitium of type 2 DN patients and db/db mice were significantly increased,especially the colocalization of macrophage with Dll4/N1 ICD was enhanced.In vitro study showed that high glucose-stimulated HK-2-derived exosomes enriched in Dll4 were internalized by macrophages and subsequently upregulated the expression of i NOS and IL-6 in macrophages,polarizing macrophages towards M1 phenotype.3.The expression of Epsin1 in the urinary exosomes and renal tubules of type 2 DN patients and db/db mice was increased and was positively correlated with Dll4/N1 ICD and tubulointerstitial damage score(IFTA).After the injection of HG-stimulated HK-2-derived exosomes into C57BL/6 mice,their serum BUN and Scr levels were significantly increased.Renal tissue damage and increased inflammatory factors(IL-6,TNF-α),macrophage marker proteins(F4/80,i NOS),Dll4/N1 ICD were also observed.Whereas,Epsin1 KD abolished these effects.In vitro study showed that the content of Dll4 in exosomes produced by Epsin1 KD-HK-2 cells was decreased significantly.Similarly,despite both Dll4-OE transfection and HG treatment,Epsin1 KD blocked the sorting of Dll4 into HK-2-derived exosomes.THP-1macrophages receiving Dll4 OE-HK-2-derived exosomes upregulated the expression of i NOS,Dll4 and N1 ICD,while Epsin1 KD-HK-2-derived exosomes could not induce these increasements.Conclusions:High glucose promotes TEC-Dll4 sorting into exosomes,which can target macrophages and induce macrophages towards M1 phenotype by activating Notch1 signaling pathway,thus promoting renal tubulointerstitial inflammation.This novel mechanism plays an important role in the DN progression.More importantly,this sorting of Dll4 into exosomes is Epsin1-dependent.Epsin1 inhibition can alleviate Dll4-encapsulated exosome-induced renal damage.