Fibroblast-Selective Smoothened Sensing of the Fibrotic Microenvironment by Controlling Energy Metabolism

Background: Smoothened (Smo) is a serpentine transmembrane protein that regulates hedgehog signaling through an intracellular conformational switch controlled by electrostatic interactions and multiple phosphorylations. Our previous study found that the loss of fibroblast-Smo decreased tyrosine phosphorylation on GSK-3β, activating the Wnt signaling pathway to mitigate acute kidney injury. However, the role of fibroblast-selective Smo in kidney fibrosis remains unclear. Methods: We generated two strains of inducible fibroblast-specific Smo conditional knockout (cKO) mice using Gli1-Cre and Pdgfrβ-Cre drivers. These mice underwent unilateral renal ischemic reperfusion injury combined with nephrectomy (UIRI [10 days] + Nx [7 days]) or unilateral ureter obstruction (UUO [7 days]) to induce kidney fibrosis. Global, phospho-, and spatial-proteomics were employed to analyze the results. Results: Following UIRI + Nx or UUO, both strains of fibroblast-Smo cKO mice showed improved renal function and reduced kidney fibrosis. Global proteomics identified 5,945 differential candidates out of 8,024 proteins in fibrotic kidneys. Among these, 67 significant core-matrisome proteins were highlighted, with fibulin-2 being the most notably altered. Phosphoproteomics identified 10,740 phospho-sites corresponding to 3,351 non-redundant phosphoproteins. KEGG enrichment analysis indicated that the epidermal growth factor (EGF) signaling pathway was inactivated in cKO mice compared to controls. Notably, the phosphorylation of most EGF downstream proteins was substantially reduced at multiple serine and tyrosine sites. Mechanistically, fibroblast-released fibulin-2 directly interacted with tubular EGFR, causing EGFR phosphorylation and mitochondrial translocation. This suborganelle translocation induced acetyl-CoA acetyltransferase 1 (ACAT1), as confirmed by spatial proteome analysis. Consistent with Gene Ontology analysis of the global- and phospho-proteome showed upregulated fatty acid β-oxidation and amino acid metabolism in cKO kidneys, as a mitochondrially localized enzyme, ACAT1 indeed processed amino acids and fatty acids in energy metabolism, facilitating the alleviation of kidney fibrosis in fibroblast-Smo cKO mice. Conclusion: Fibroblast-specific deletion of Smo reduced EGFR phosphorylation and its mitochondrial translocation, improving the fibrotic kidney microenvironment. Funding: NIDDK Support