Figure 5

KRM promote proliferation of PLVAP⁺CD31⁺ renal peri-tubular endothelial cells and inhibit TGF-β-induced expression of Col1a in vitro. (A) Co-culture of RAS-KRM with PLVAP⁺CD31⁺ endothelial cells. EdU incorporation between BM macrophages (Mϕ), RAS-KRM and Sham-KRM compared to FOXO1-inhibitor AS1842856. (B) CellTrace Far Red dye dilution assay of PLVAP⁺CD31⁺ cells in a contact co-culture system. (C) TGF-β induces dose-dependent expression of Col1a in murine embryonic fibroblasts (MEF) derived from Col1-GFP mice (Lane1–3, Left and right graph). The increase in Col1a was inhibited by UO126 (MEK inhibitor) and LY2109761 (TGF-β receptor I and II dual inhibitors) (Left). Co-incubation of Sham-KRM (Lane5) and RAS-KRM (Lane6) with MEF (GFP) significantly inhibited the increase in Col1a1-GFP (Right), while BMϕ (Lane7) and CD11c^hi/loMϕ (Mϕ1,2) (Lane8) had no effect. BMϕ = bone marrow macrophages; Mϕ1,2 are (n = 5 technical replicates and n = 3 biological replicates per sample); (D) Representative images demonstrating contact co-culture of Col1a1-GFP MEFs (green) with (bottom) and without (top) KRM stained with anti-mouse CD64-AF594 (red) *P < 0.01 vs MEF untreated control; ^§P < 0.01 by One-sample T-test; ^†P < 0.05 vs Control.