Increased Smoothened signaling promotes kidney fibrosis through inhibiting autophagy in fibroblasts.

Abstract

Kidney fibrosis progressively induces kidney function loss, leading to kidney failure. Myofibroblast transition from fibroblast is significantly involved in the development of kidney fibrosis. Previous studies revealed that sonic hedgehog (Shh), the upstream ligand of Smoothened (Smo) receptor signaling, contributes to kidney fibrosis. However, the role of Smo in fibroblast activation remains unclear. Here, fibroblast-specific Smo and β-catenin knockout mice were generated, primary kidney fibroblasts were isolated and cultured. Single-nucleus (snRNA-seq) plus bulk RNA sequencing were performed. Autophagy capacities, including autophagy flux, autophagy vacuoles, and autophagy-related gene expression, were assessed. From snRNA-seq data of the human atlas combined with bulk RNA sequencing of primary mouse kidney fibroblasts, we discovered Smo was upregulated in kidney fibroblasts in chronic kidney disease (ureteral obstruction, ischemia/reperfusion, Adriamycin nephrosis and 5/6th nephrectomy models). This was accompanied by substantially diminished autophagic capacity. Shh stimulation or ectopic Smo deactivated autophagy, exaggerated myofibroblast proliferation and activation in the fibroblasts. These effects were further enhanced in autophagosome protein ATG5 knockout fibroblasts. Moreover, Smo, through the β-arrestin 1/Src/β-catenin pathway, induced the transcription of mammalian target of rapamycin. This mediated autophagy suppression in fibroblasts, leading to myofibroblast activation and kidney fibrosis. Fibroblast-specific knockout of Smo or β-catenin greatly preserves autophagy capacity and ameliorates kidney fibrosis. Notably, NVP-LDE225, a specific Smo antagonist, effectively restored fibroblast autophagy and retarded kidney fibrotic lesion formation. Our study defines a prominent mechanism of myofibroblast activation and supplies a potential avenue for targeting fibroblast Smo to treat chronic kidney disease.