Gene Signature-guided Drug Screening Identified Narciclasine as a Potential Therapeutic Drug for Renal Interstitial Fibrosis

Chronic Kidney Disease (CKD) is characterized by progressive tubulointerstitial fibrosis (TIF), a condition not adequately addressed by existing therapies. To identify potential compounds that could slow or halt the progression of TIF, we established gene signatures of TIF using published human CKD renal transcriptome data, followed by high-throughput screening of compounds that could reverse TIF-associated gene expression using the LINCS L1000 database. The natural compound Narciclasine (Ncls), extracted from daffodils, was identified as a promising candidate. We validated the effects of Ncls on a TGF-ß1-induced fibrosis model in vitro using mouse primary tubular epithelial cells (TECs) and rat fibroblast cell line (NRK49F). Results demonstrated that Ncls significantly inhibited the pro-inflammatory and pro-fibrotic effects induced by TGF-ß1 in TECs. Additionally, in fibroblasts, Ncls markedly suppressed the proliferation and activation induced by TGF-ß1. These findings not only confirm the therapeutic potential of Ncls in managing TIF but also highlight the efficacy of gene signature-based screening in identifying drugs for the treatment of CKD. Overall design: We utilized isolated mouse primary tubular epithelial cells (TECs) and the rat kidney fibroblast cell line (NRK49F) to validate the effects of Narciclasine (Ncls) on TGF-ß1-induced pro-fibrotic responses in vitro. Ncls was diluted to a final concentration of 20 nM in serum-free DMEM/F12 medium. TECs were pre-incubated in this solution for 2 hours. Subsequently, TGF-ß1 was added to achieve a concentration of 5 ng/ml in the medium, and the cells were co-stimulated with Ncls for 24 hours. The control group contained only the medium, without the addition of TGF-ß1 or Ncls. Cell samples were then collected for RNA-seq analysis. Experimental samples were organized into three groups with three biological replicates each: Control TECs (Con_TECs), TECs stimulated with TGF-ß1 alone (Tgfb_TECs), and TECs co-stimulated with TGF-ß1 and Ncls (T+N_TECs). The experimental procedure for NRK49F cells mirrored that of the TECs. Experimental samples for NRK49F were also divided into three groups with three biological replicates each: Control 49F (Con_49F), 49F cells stimulated with TGF-ß1 alone (Tgfb_49F), and 49F cells co-stimulated with TGF-ß1 and Ncls (T+N_49F).