Phase separation mediated transcriptional activity of FOXK1 triggers glycolysis and facilitates renal fibrosis
Tubular epithelial cells (TECs) undergo an energy metabolism shift from fatty acid oxidation (FAO) to glycolysis in renal fibrosis. The critical pathways leading to the halt of FAO in TECs have been well described, whereas the mechanism underlying the burst of glycolysis remains elusive. We herein reported a critical glycolysis regulator emerged in TECs amid the renal fibrosis, the transcriptional factor forkhead box protein K1 (FOXK1), which exhibited fibrogenic and metabolism-rewiring capacity. Genetic modification of FOXK1 in TECs altered the glycolytic metabolism and fibrotic lesion. The surge of a set of glycolysis-related genes following FOXK1 activation contributed to the energic shift. Nuclear-translocated FOXK1 formed condensates through liquid-liquid phase separation (LLPS) to drive the target genes transcription. The core intrinsically disordered regions (IDRs) within FOXK1 were further mapped and validated. Finally, we explored the therapeutic strategy targeting Foxk1 in the CKD mouse model by subcapsular injecting Foxk1-shRNA-carrying AAV9 vector. Overall design: To explore the transcriptional regulatory mechanisms underlying the roles of FOXK1 in renal fibrosis, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze the genome-wide distribution of FOXK1 in HK-2 cells. Cells were cultured in DMEM/F12 medium with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37 °C incubator with 5% CO2. Cells were seeded on six-well culture plates for transfection and treatment with or without 10 ng/ml TGF-ß1 for 24 hours.