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 3

Global proteomics identifies NID1 as a prominent matrix protein in Gli1+ fibroblast-specific Smo deletion kidneys after AKI.

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Global proteomics identifies NID1 as a prominent matrix protein in Gli1+...
(A) Experimental workflow of the global proteomic analysis. 6 mice in each group were used for mass spectrometry. (B) Principal component analysis of global proteomes from Gli1-Smo+/+ and Gli1-Smo–/– kidneys 1 day after ischemic AKI. (C) Heatmap of ANOVA-significant proteins. Label-free quantitation (LFQ) intensity of represented proteins were z-scored and plotted according to the color bar. 2 clusters of proteins with different patterns of abundance profiles are highlighted in the heatmap. (D) GO cellular compartment terms in each cluster of proteins are plotted with their names and significance. ECM proteins are boxed to indicate the protein group with the largest difference in upregulated proteins. (E) Volcano plot shows the differential proteins between Gli1-Smo+/+ and Gli1-Smo–/– kidneys. Up and down regulated proteins (FC, fold-change) are colored in red and blue, respectively. Yellow dots indicate ECM proteins. (F) Heatmap of differentially expressed ECM proteins. (G and H) Western blots of NID1 protein in Gli1-Smo+/+ and Gli1-Smo–/– or in Pdgfr-β-Smo+/+ and Pdgfr-β-Smo–/– kidneys 1 day after IRI. Numbers indicate individual animals in a given group. (I) Single nucleus RNA-Seq showing NID1 mainly expressed by fibroblasts (Fib) and pericytes (Per) 12 hours after IRI. (Data were extracted from the online database provided by Benjamin Humphrey’s laboratory, http://humphreyslab.com/SingleCell/displaycharts.php) (J) IHC staining showing NID1 protein expression in Gli1-Smo+/+ and Gli1-Smo–/– or in Pdgfr-β-Smo+/+ and Pdgfr-β-Smo–/– kidneys 1 day after IRI. Arrows indicate positive staining. Scale bar: 25 μm. (K) Immunoprecipitation revealing that Smo binds to NID1. NRK-49F cells under CoCl2 stress were immunoprecipitated with Smo or NID1 antibody, followed by immunoblotting with antibody against NID1 (left blot) or Smo (right blot). Differences among groups were analyzed using unpaired t tests.