Krüppel-Like Factor 6 Induces RNA Polymerase II Subunit RPB1 to Promote Kidney Injury.

Abstract

Single nuclear RNA sequencing after DNA damage–induced AKI identified an injured proximal tubule cluster with high <i>Polr2a</i> (RNA polymerase subunit B1), an RNA polymerase II subunit. <i>POLR2A</i> knockdown in injured cells decreased inflammatory and fibrotic gene expression, dedifferentiation, DNA damage, and cell cycle arrest. RNA polymerase subunit B1 was higher in mice overexpressing transcription factor Krüppel-like factor 6 and associated with worse injury after DNA damage. Initial proximal tubule cell injury and dedifferentiation contribute to AKI, and persistent dedifferentiation drives fibrosis and CKD. Proximal tubule–specific knockdown of zinc-finger transcription factor <i>Krüppel-like factor 6</i> (<i>Klf6</i>) attenuates the AKI to CKD transition. Our aim was to study the early transcriptional mechanisms by which KLF6 induction exacerbates proximal tubular injury and eventual fibrosis. Aristolochic acid I–treated wild-type and KLF6 overexpression mice underwent single nucleus (sn)RNA-seq and single nucleus assay for transposase-accessible chromatin sequencing (acute phase) and assessment of kidney function, injury, and fibrosis (remodeling phase). <i>POLR2A</i> was knocked down in human kidney cells and cell number, gene expression, differentiation, DNA damage, and cell cycle assessed. Kidney sections from fibrotic mouse models and human CKD secondary to aristolochic acid and diabetes were assessed for RNA polymerase subunit B1 (RPB1) expression. snRNA-seq identified an injured proximal tubule cluster with high expression of <i>Klf6</i> and RNA polymerase II subunit a (<i>Polr2a</i>) encoding RPB1. After injury, RPB1-positive cells accumulated and were associated with dedifferentiated proximal tubules. <i>POLR2A</i> knockdown in injured cells increased cell death, but reduced inflammatory and fibrotic gene expression, dedifferentiation, DNA damage, and G2/M cell cycle arrest, with a transcriptional switch from long genes to short genes. Single nucleus assay for transposase-accessible chromatin sequencing demonstrated an open chromatin region in <i>Polr2a</i> intron 1 in injured proximal tubule cells, containing a KLF6-binding site. Knockdown of <i>KLF6</i> reduced <i>POLR2A</i> induction, while proximal tubule–specific <i>KLF6</i> further increased <i>Polr2a</i> levels after injury. Mice with tubule-specific <i>KLF6</i> induction had more RPB1-positive proximal tubules and more injury post-AKI. Human kidney samples with DNA damage–induced CKD and diabetic kidney disease also had high <i>POLR2A</i>/RPB1 expression in dedifferentiated proximal tubule cells. Prolonged high expression of RPB1 is associated with dedifferentiated proximal tubule cells. Mice overexpressing <i>KLF6</i> had higher expression of RPB1 and worse kidney injury after DNA damage.