Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury
Yuan Gui, … , Youhua Liu, Dong Zhou
Yuan Gui, … , Youhua Liu, Dong Zhou
Published May 7, 2024
Citation Information: J Clin Invest. 2024;134(13):e165836. https://doi.org/10.1172/JCI165836.
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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 7

NID1 promotes Wnt components expression and reduces tubular cell death in vitro.

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NID1 promotes Wnt components expression and reduces tubular cell death i...
(A–D) Normal rat kidney fibroblasts (NRK-49F) were transfected with Dicer-substrate Smo siRNA (Smo Dsi) or incubated with Smo inhibitor cyclopamine (CPN, 2.5 μM), then subjected to hypoxic stress (CoCl2, 400 μM) for 24 hours. Compared to control siRNA (NC Dsi) or vehicles, western blots demonstrating that knockdown (A) or inhibition (B) of Smo induced Pdgfr-β, vimentin, αSMA, and PCNA expression, and increased NID1 (C and D). (E) Immunofluorescence staining showing Smo inhibition induced NID1 in fibroblasts under hypoxic stress. Scale bar: 25 μm. Arrows indicate positive staining. (F) Enzyme-linked immunosorbent assay revealed NID1 concentration after incubation with CPN under hypoxic stress (n = 6). (G) Schematic diagram. (H and I) Western blots demonstrating β-catenin, Wnt1, Wnt2, and Wnt5A/B levels after knockdown (H) or inhibition (I) of Smo under hypoxic stress. (J and K) Western blots showing that NID1 recombinant protein (rNID1) elevated β-catenin, Wnt1, Wnt5A/B, and Wnt16 in cultured normal rat kidney proximal tubular cells at different dosages under basal conditions (J) and hypoxic stress (K). (L, M, and O) After stimulation with staurosporine (1 μM) for 3 h, western blots assay showing reduced cleaved-caspase 3 (CCP3) in tubular cells incubated with NID1-enriched CM collected from Smo-knockdown (L) or CPN-treated (M) fibroblasts or directly treated with rNID1 (O). (N and P) Immunofluorescence staining showed fewer CCP3+ cells after treated with NID1-enriched CM (N) or NID1 recombinant protein (P). Quantitative data are presented in Q (n = 3, 4 random images were selected per slide, each dot represents the score of the according image). †P < 0.05 versus control, *P < 0.05 versus vehicle after STS. Scale bar: 25 μm. Cells were costained with DAPI. Arrows indicate positive staining. Graphs are presented as means ± SEM. Differences among groups were analyzed using 1-way ANOVA, followed by the Student-Newman-Keuls test.