Fig. 8. Integrin β1 is required for FXII-uPAR signaling on tubular cells.

a Volcano plot comparing hyperglycemic F12^-/- mice to hyperglycemic WT mice based on Log fold change (FC) values and the false discovery rate (FDR); integrins are shown in green. Integrin β1 (Itgb1) is the most downregulated integrin in hyperglycemic F12^-/- mice kidneys by FDR. b Bar graphs summarizing the expression (qRT-PCR) of selected integrin genes comparing normoglycemic controls and hyperglycemic WT and F12^-/- mice. Bar graphs reflecting mean ± SEM of 4 mice per group; two-way ANOVA with Tukeys’s multiple comparison test. c Representative immunoblots for active integrin β1, active integrin β3, and uPAR from uPAR coimmunoprecipitation (IP, top) and immunoblots for FXII, uPAR, and α-Tubulin from the input (input, bottom,) of HKC-8 cells exposed to purified human FXII (62 nM) in the presence of Zn²⁺ (10 µM) for 24 h (FXII) compared to control non-treated cells (C). Input serves as a loading control. Immunoblots represent 3 independent experiments. d, e Representative images of proximity ligation assay (PLA, d; red, uPAR and active integrin β1 interaction) and dot-plot summarizing results (e) in experimental groups (as described in c); DAPI nuclear counterstain, blue; phalloidin for cytoskeleton, green. Scale bars represent 20 μm. Dot-plot reflecting mean ± SEM of 3 independent experiments quantifying 30 cells from each condition with each dot representing the number of PLA signals/cell; two-tailed unpaired student’s t test. f, g Representative images of proximity ligation assay (PLA, f; red dots representing uPAR and active integrin β1 interaction) and dot-plot summarizing results (g) in F12-null HKC-8 cells transfected with wild type FXII (WT-FXII) or a FXII deletion mutant lacking fibronectin type II domain (∆Fib-II-FXII) compared to empty-vector transfected cells (C, controls); DAPI nuclear counterstain, blue; and phalloidin for cytoskeleton, green. Scale bars represent 20 μm. Dot-plot reflecting mean ± SEM of 3 independent experiments quantifying 30 cells from each condition with each dot representing the number of PLA signals/cell; one-way ANOVA with Tukeys’s multiple comparison test. h Representative images of proximity ligation assay (PLA, f; red dots representing uPAR and active integrin β1 interaction) in human kidney sections of nondiabetic controls (C) or diabetic patients with DKD (DKD); DAPI nuclear counterstain, blue; and FXII, green. Scale bars represent 20 μm. Source data are provided as a “Source Data” file.