Although TGF-1 signaling through the Smad-based (canonical) pathway5C7 is believed to play a critical role in the development of renal fibrosis, a growing body of evidence indicates that several non-Smad (non-canonical) pathways stimulated by TGF-1 are also potentially involved in driving fibrosis in progressive kidney disease8C10. of -SMA, fibronectin, phospho-JNK, and cleaved Notch-2; however, the levels of phospho-Smad2/3, phospho-p38 and phospho-ERK remained unchanged. Pharmacological inhibition of different signaling pathways and genetic knockdown of Notch-2 further revealed JNK as an upstream effector of Notch-2 in TGF-1-mediated IWP-O1 renal fibrosis. Consistently, we also demonstrated that administration of TSA or a -secretase inhibitor RO4929097 in the mouse model of unilateral ureteral obstruction significantly ameliorated renal fibrosis through suppression of the JNK/Notch-2 signaling activation. Taken together, our findings provide further insights into the crosstalk among different signaling pathways in renal fibrosis, and elucidate the molecular action of TSA in attenuating fibrogenesis. Introduction Renal fibrosis is the final pathological process common to all forms of chronic kidney disease1 and thereby represents an excellent treatment target. It is characterized by accumulation and activation of myofibroblasts, and extensive deposition of extracellular matrix in kidney parenchyma2. During development of renal fibrosis2,3, transforming growth factor-1 (TGF-1) is considered as the master mediator that induces myofibroblastic activation4 and abundant deposition of fibrotic matrix in renal tubulointerstitium. Although TGF-1 signaling through the Smad-based (canonical) pathway5C7 is believed to play a critical role in the development of renal fibrosis, a growing body of evidence indicates that several non-Smad (non-canonical) pathways stimulated by TGF-1 are also potentially involved in driving fibrosis in progressive kidney disease8C10. Among these TGF-1-induced non-Smad signaling pathways, three major mitogen-activated protein Jun kinases (MAPKs) pathways (including p38, ERK and JNK) have been suggested to contribute to inflammatory and fibrotic damages of various renal diseases11C13. Thus, detailed understanding the downstream networks of TGF-1-mediated signaling during the progression of renal fibrosis would be helpful to develop new therapeutic strategies to prevent or delay kidney damage. The Notch signaling pathway is an evolutionarily conserved pathway, which is known to play an essential role in renal development14. After completion of renal development, the Notch signaling pathway is largely suppressed15. In vertebrates, the Notch system consists of four highly conserved membrane receptors (Notch-1 to Notch-4) and five ligands (JAG-1, JAG-2, Delta-like-1, Delta-like-3, and Delta-like-4). Activation of Notch signaling pathway is initiated through the binding of ligands to Notch receptors. Upon ligand binding, the Notch receptor undergoes two consecutive proteolytic cleavages by ADAM metalloprotease and -secretase, ultimately leading to the release of IWP-O1 the Notch intracellular domain (NICD). The resultant NICD then translocates into the nucleus, where it interacts with RBP-J (also known as CSL or CBF-1) and Mastermind like-1 coactivator to cooperatively activate its downstream target genes, such as hairy enhancer of split (Hes) and Hes-related repressor (Hey) families16. Emerging evidence has shown that aberrant activation of the Notch signaling pathway could lead to epithelial-mesenchymal transition (EMT) and regulate interstitial fibrosis17,18. Murea and and model of renal fibrosis, NRK-49F cells, a rat kidney interstitial fibroblast cell line, were first treated with increasing amounts of TGF-1 (0, IWP-O1 1, 2 and 5?ng/ml) for 48?h. Western blot analysis showed that TGF-1 at the dose of 5?ng/ml substantially increased levels of -SMA and fibronectin, two hallmarks of activated fibroblasts, in treated cells (Supplementary Fig.?S1a), and thus this concentration of TGF-1 was suitable for subsequent experiments. Additionally, in time-course experiments, we found that the maximal induction of -SMA was reached at 48 hr after treatment with TGF-1 at 5?ng/ml (Supplementary Fig.?S1b). To further examine the activation of TGF-1-mediated canonical or non-canonical signaling pathways in NRK-49F cells, short-term treatment of cells with TGF-1 was performed. Western blot analysis revealed that TGF-1 treatment rapidly induced phosphorylation of Smad2 and Smad3 in NRK-49F cells, and the levels of phospho-Smad2 and phospho-Smad3 reached a maximum at 30 and 60?min post-treatment, respectively (Supplementary Fig.?S1c). Furthermore, TGF-1 treatment also significantly increased the levels of phospho-p38, phospho-ERK and phospho-JNK in these treated cells, which reached a maximum at 90?min post-treatment.