Background: Kidney fibrosis is a major clinical problem, and a final common process of injury. Angiotensin II (AngII), the main effector of the renin-angiotensin system (RAS) mediates native and allograft kidney fibrosis. Using SILAC-based proteomics, we previously identified AngII-regulated proteins in proximal tubular cells (PTECs) that may play a role in fibrosis in vivo. Selected Reaction Monitoring (SRM) assays targeting 6 AngII–regulated proteins demonstrated significant increase in urine excretion of these proteins in patients with kidney graft fibrosis compared to no fibrosis. The purpose of this study is to investigate how our fibrotic proteins are regulated using chromatin accessibility profiling and transcription factor footprinting analyses.
Methods: PTECs were subjected to AngII (10-7M) or vehicle and analyzed by ATAC-seq. Purified nuclei were incubated with Tn5 transposase loaded with adapters, purified and amplified with PCR. The amplified libraries were multiplexed and sequenced. The analysis of sequencing data involved aligning of ATAC-seq peaks over samples, quality checking, intensity normalization and short-listing of the replicable ATAC-seq regions differentially open between AngII and vehicle-treated PTECs.
Results: We identified 82 regions of differentially open chromatin between AngII-stimulated and vehicle-treated PTECs (p<0.05) that were accessible in kidney tissue. By using GTRD transcription factor database, we discovered 20 transcription factors with the highest frequency of binding to our differentially accessible ATAC-seq regions. Five transcription factors (YY1, RUNX1, SP1, CTCF, AR) have been previously linked to kidney fibrosis, AngII or chronic kidney disease. GREAT pathway analysis confirmed that these differentially accessible ATAC-seq regions were enriched in fibrotic pathways such as TGFb, SMAD2/3 signaling, and activation of the AngII receptor.
Conclusions: Using ATAC-seq, we identified open chromatin regions and transcription factors in PTECs stimulated with AngII. We confirmed that these regions were enriched in fibrotic signaling pathways. Further studies will aim to link five transcription factors to AngII-regulated proteins.