Abstract: FR-PO344

Dickkopf-3 (Dkk-3) Overexpressed in Dysfunctional Endothelium Secretome Instructs Fibroblast-to-Myofibroblast Formation by Activating the Wnt Pathway

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

  • Lipphardt, Mark, New York Medical College, Valhalla, United States
  • Dadafarin, Sina, New York Medical College, Valhalla, New York, United States
  • Ratliff, Brian B., New York Medical College, Valhalla, New York, United States
  • Dihazi, Hassan, University Medical Centre Goettingen, Goettingen, Germany
  • Mueller, Gerhard A., Georg-August University, Gottingen, Germany
  • Goligorsky, Michael S., New York Medical College, Valhalla, New York, United States
Background

We have previously demonstrated that Sirt1endo-/- mice with endothelial dysfunction show exaggerated renal fibrosis, whereas mice with silenced endothelial TGF-β signaling are resistant to fibrogenic signals. Collectively, this indicates that secreted substances regulate these contrasting responses. Thus, we sought to examine the differential secretome of those cells.

Methods

We performed unbiased proteomic analysis of the secretome of renal microvascular endothelial cells (RMVEC) isolated from these two mutants, discovered Dkk-3 (a putative ligand of Wnt/β-catenin pathway) expressed exclusively in fibrogenic secretome. Since Dkk-3 is an orphan member of the family of Wnt ligands, and its precise effects are poorly understood, we examined effects of Dkk-3 in renal fibroblasts (RF) isolated from α-SMA-GFP mouse kidneys using positive and negative selection with magnetic beads.

Results

Application of Dkk3 to RF showed that Dkk3 (10 ug/ml) alone induced myofibroblastic phenotype without altering responses to TGFβ. Dkk-1, a known antagonist of Wnt pathway, reduced activation of RF. When Dkk-3 was combined with Dkk-1, it antagonized its antimyofibroblastic effect. In RMVEC, Dkk-3 induced endothelial-mesenchymal transition (endo-MT) as judged by the appearance of α-SMA-GFP signal, and reduced capillary cords formation and their branching angiogenesis. In microfluidic RF-RMVEC co-cultures (kindly provided by NL Jeon, Seoul National University, Seoul, Korea) Dkk-3 was confirmed as an inducer of endo-MT and inhibitor of angiogenesis. Chronic administration of Sulindac, a potent Wnt pathway inhibitor, ameliorated UUO-induced renal fibrosis.

Conclusion

In conclusion, a prominent member of the secretome of dysfunctional RMVEC, Dkk-3, affords a Dkk-1 antagonistic paracrine effect on RF and induces myofibroblastic prenotype. Dkk-3 exerts a autocrine effect leading to endo-MT of RMVEC and reducing their angiogenic competence. These actions make Dkk-3 a potent pro-fibrogenic agonist.