Abstract: SA-OR099

Indoxyl sulfate Promotes Macrophage Activation via Novel Crosstalk between OATP2B1 and Dll4-Notch Signaling

Session Information

Category: Hypertension

  • 1103 Vascular Biology and Dysfunction

Authors

  • Nakano, Toshiaki, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Decano, Julius L., Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Katsuki, Shunsuke, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Boff, Mario S, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Irvin, Whitney S, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Higashi, Hideyuki, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Singh, Sasha, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Aikawa, Elena, Brigham and Women''s Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Aikawa, Masanori, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
Background

CKD increases cardiovascular risk, however, its underlying mechanisms remain obscure. We previously reported the role of the Notch signaling ligand Delta-like 4 (Dll4) in macrophages. We tested the novel hypothesis that Dll4-Notch mediates macrophage activation stimulated by indoxyl sulfate.

Methods

We examined the effects of indoxyl sulfate (0.25–1.0 mM) on pro-inflammatory responses and Notch signaling in macrophages and the roles of organic anion transporters / polypeptides (OATs/OATPs). To determine the contribution of OATP2B1 to pro-inflammatory activation of macrophages in vivo, we used macrophage-targeted lipid nanoparticles to deliver siRNA to mice. To address the role of Dll4 in atherogenesis in CKD, we used 5/6 nephrectomy and Dll4 neutralizing antibody in mice.

Results

[In vitro] In human primary macrophages, indoxyl sulfate induced pro-inflammatory molecules IL-1β, TNFα, and MCP-1 and Notch signaling components Dll4, Notch1 and Hes1. Decreased degradation by ubiquitin-proteasome pathway appears to promote rapid induction of Dll4 < 1 hour by indoxyl sulfate (FACS, Western blotting, immunofuorescence), which in turn triggers Notch signaling. Notch inhibition with the γ-Secretase inhibitor, Dll4 siRNA or Dll4 antibody indeed suppressed indoxyl sulfate-induced macrophage activation. We identified previously unreported 9 OATs/OATPs in macrophages, among which human and mouse commonly expressed OATP2B1. Suppression of OATP2B1 suppressed macrophage uptake of indoxyl sulfate (HPLC), Notch signal components Dll4 and Hes1, and pro-inflammatory genes. [In vivo] Indoxyl sulfate administration in mice promoted expression of IL-1β and MCP-1 in peritoneal macrophages. Co-administration of Dll4 antibody or OATP2B1 siRNA encapsulated in macrophage-targeted nanoparticles suppressed these responses (p<0.05, n=6-8). In LDL receptor-deficient mice, 5/6 nephrectomy enhanced Notch signaling and the development of atherosclerosis, macrophage burden, calcification, and pro-inflammatory responses, which were abrogated by Dll4 antibody (p<0.01 vs. IgG; n=20/group).

Conclusion

These lines of in vitro and in vivo evidence suggest that crosstalk between OATP2B1 and Dll4-Notch signaling mediates indoxyl sulfate-induced macrophage activation and vascular disease.