Injured Epithelia with Nuclear Factor kB Activation Drives Renal Fibrosis by Remodeling the Fibrotic Microenvironment

Background: Tubular epithelial injury significantly influences chronic kidney disease (CKD) progression. A subset of injured proximal tubule cells (iPT), characterized by markers Vcam1 and Kim1, has been observed in both acute and chronic kidney diseases and appears to play a crucial role in promoting fibrosis by remodeling the renal microenvironment. However, the transcription factors driving iPT-mediated fibroinflammatory reprogramming remain unclear. Methods: We integrated cross-species single-cell and spatial transcriptomic datasets from human, rat, and mouse kidneys to identify conserved signaling pathways in iPT cells. CUT&RUN profiling of four key NF-κB subunits (NF-κB1, RelA, NF-κB2, RelB) was performed on primary tubular cells. Functional validation was conducted in RPTEC cultures stimulated with injury-related cytokines (e.g., IFN-γ/TNF-α), followed by pharmacologic inhibition and CRISPR interference. The specific contribution of each subunit was assessed using Sox9+ -targeted conditional knockout models (Sox9CreERT2-Nfkb1f/f, -RelAf/f, -Nfkb2f/f, -RelBf/f) subjected to adenine-induced CKD. Results: NF-κB pathway activation emerged as a conserved transcriptional signature in iPT cells across species. In vitro, IFN-γ/TNF-α synergistically induced NF-κB activation and a profibrotic-inflammatory phenotype mimicking iPT cells. Integrated CUT&RUN, scRNA-seq, and scATAC-seq analyses identified the NF-κB2/RelB axis as most closely associated with the iPT transcriptional program. Among the four NF-κB subunits, conditional deletion of Nfkb2 in Sox9+ tubular cells most effectively reduced iPT cell expansion and mitigated fibroinflammation, outperforming RelA and RelB deletion. Further analysis revealed that NF-κB2 orchestrates a transcriptional network that promotes epithelial reprogramming and fibrotic niche remodeling. Conclusion: These findings identify NF-κB2 as the key NF-κB subunit driving fibroinflammatory reprogramming in injured tubular epithelium, highlighting its potential as a targeted therapeutic strategy in CKD. Targeting NF-κB2-driven pathways may significantly alter the trajectory of fibrosis development, offering critical insights into novel treatment approaches for kidney disease. Funding: NIDDK Support