(A) Experimental design. In step 1, normal rat kidney fibroblasts (NRK-49F) were transfected with Dicer-substrate Smo siRNA (Smo Dsi) or cultured with Smo inhibitor CPN to enrich for NID1. In step 2, scaffolds were isolated, and, in step 3, normal rat kidney proximal tubular cells (NRK-52E) were seeded on top of scaffolds. (B) Western blots assay showing NID1 protein in decellularized fibroblast matrix scaffold. (C and D) Western blots assay showing β-catenin, Wnt2, and Wnt5A/B were activated in NRK-52E cells seeded on NID1-enriched matrix scaffold isolated from Smo-knockdown (C) or CPN-treated (D) fibroblasts under hypoxic stress. (E and F) Western blots assay showing NID1-enriched matrix scaffold isolated from Smo-knockdown (E) or CPN-treated (F) fibroblasts reduced cleaved caspase-3 (CCP3) in the seeded NRK-52E cells. (G–I) Western blot assay showing conditioned medium collected from Smo-knockdown fibroblasts (G) or decellularized fibroblast matrix scaffold isolated from Smo-knockdown fibroblasts (H) or NID1 recombinant protein (rNID1) increased integrin β1 in NRK-52E cells under hypoxic stress. (J and K) Under hypoxic stress, knockdown of integrin β1 using Dicer-substrate siRNA (integrin β1 Dsi) repressed β-catenin accumulation after incubation with rNID1 in NRK-52E cells (J) and induced CCP3 (K). (L) Molecular docking analysis showing the binding sites between NID1 and Integrin β1. (M) The strategy of designing a mutant form of NID1. (N and O) Compared with the active form of NID1 human recombinant protein (25 ng/mL), Western blot assay demonstrating that mutant NID1 (25 ng/mL) failed to induce β-catenin expression in NRK-52E cells under hypoxic stress (N) and increased CCP3 after staurosporine (1 μM) stimulation (O). (P) Our model illustrates that loss of fibroblast-selective Smo promotes NID1 to interact with tubular integrin β1 and subsequently activated the Wnt signaling pathway in tubules, forming a favorable microenvironment to protect against AKI.