Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury
Yuan Gui, Haiyan Fu, Zachary Palanza, Jianling Tao, Yi-Han Lin, Wenjian Min, Yi Qiao, Christopher Bonin, Geneva Hargis, Yuanyuan Wang, Peng Yang, Donald L. Kreutzer, Yanlin Wang, Yansheng Liu, Yanbao Yu, Youhua Liu, Dong Zhou
Yuan Gui, Haiyan Fu, Zachary Palanza, Jianling Tao, Yi-Han Lin, Wenjian Min, Yi Qiao, Christopher Bonin, Geneva Hargis, Yuanyuan Wang, Peng Yang, Donald L. Kreutzer, Yanlin Wang, Yansheng Liu, Yanbao Yu, Youhua Liu, Dong Zhou
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Research Article Nephrology

Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury

Abstract

The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cell activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could affect nidogen-1 accessibility. Phosphoproteomics revealed that the ‘AKI protector’ Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin β1 to induce Wnt in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.

Authors

Yuan Gui, Haiyan Fu, Zachary Palanza, Jianling Tao, Yi-Han Lin, Wenjian Min, Yi Qiao, Christopher Bonin, Geneva Hargis, Yuanyuan Wang, Peng Yang, Donald L. Kreutzer, Yanlin Wang, Yansheng Liu, Yanbao Yu, Youhua Liu, Dong Zhou

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Figure 8

NID1-enriched decellularized fibroblast matrix scaffold activates Wnt components and reduces tubular cell death ex vivo.

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NID1-enriched decellularized fibroblast matrix scaffold activates Wnt co...
(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.