RNA Polymerase II Subunit POLR2A/RPB1 Upregulation Is Detrimental in Kidney Injury

Background: Proximal tubule (PT) injury is a major factor in the severity of acute kidney injury (AKI) and transition to fibrosis. Our single nuclear RNA-sequencing (snRNA-seq) in a PT-specific nephrotoxin [aristolochic acid I (AAI)]-induced AKI in mice revealed an injured PT cluster with high enrichment of Polr2a, encoding RPB1, the largest subunit of RNA polymerase II (RNAPII). Our aim was to study the role of PT-specific RPB1in AKI. Methods: Immunofluorescence (IF) for RPB1 was undertaken in mice after multiple 2mg/kg AAI injections, 7mg/kg repeated low dose cisplatin (RLDC) for 4 weeks, or unilateral ureteral obstruction (UUO) for 3 or 7 days, or in human AA-exposed chronic kidney disease (CKD) patient samples, with PT brush border counterstaining. HK-2 cells were treated with DMSO or 25µM AAI and scrambled or POLR2A siRNA, with quantification of cell number (CyQuant assay); IL6, CTGF, and CDKN1A mRNA expression; and IF for vimentin (dedifferentiation) or gH2A.X (DNA damage) at 48 hours. Single cell ATAC-seq was undertaken in mice after one dose of AAI. Results: Over a series of 4 AAI injections, despite PT loss, RPB1-positive (RPB1+) tubular nuclei accumulated, and were associated with dedifferentiated PT. This was replicated in mice after RLDC and UUO. Knockdown of POLR2A in HK-2 cells treated with AAI resulted in increased cell death, but the remaining cells expressed less IL6 and CTGF, were less dedifferentiated, had less DNA damage, and more normal cell cycle, with reduced CDKN1A expression. ATAC-seq indicated a new region of open chromatin in Polr2a intron 1 in the injured PT cluster, containing AP-1 and Krüppel-like factor 6 (KLF6) transcription factor binding sites. Trajectory and RNA velocity analysis showed that Klf6 upregulation preceded Polr2a induction, suggesting regulation of Polr2a by KLF6. RPB1 remained high in the fibrotic phase after AAI, and mice overexpressing human KLF6 had more RPB1+ nuclei than control mice. Dedifferentiated PT in human AA-exposed CKD patients also had RPB1+ nuclei. Conclusion: To date, this is first study to showthat sustained high RPB1 expression after AKI may trap PT cells in a dedifferentiated state, and clearance of cells by POLR2A knockdown may be beneficial to attenuate the progression of AKI to CKD. Funding: NIDDK Support