Advanced Sequencing of Kidney Tubule Hypoxia Regulated Epithelial‐to‐Mesenchymal Transition

Abstract

Hypoxia is a known regulator of epithelial-to-mesenchymal transition in kidney tubule cells. However, culture systems have struggled to observe these transitions at high levels to capture transcriptional signatures due to the detachment of cells from culture vessels. Using the Renal Proximal Tubule Epithelial Cell stabile cell line (RPTEC-TERT1) we developed a shear stress culture system that when exposed to hypoxia (1% oxygen) result in cell dysmorphology instead of detachment, allowing for a large number of cells to be generated for transcriptomic studies. With these cells exposed to hypoxia, we performed both traditional polyA RNAseq using both Illumina and Nanopore technologies and newer direct RNAseq using Nanopore. The polyA RNAseq suggests regulation changes for HIF-1/hypoxia, Nonsense mediated decay, ruffle assembly, and energetic regulated genes. Direct RNAseq, a strategy of running mRNA molecules across Nanopore to detect RNA base modifications transcriptome wide, revealed genes that are acutely modified related to glycolysis, innate immunity, oxidation-reduction, and mitochondrial biology. Cells exposed to hypoxia for 48 hours followed by 48 hours of normoxia, return the modifications back to control levels suggesting there is a major source of hypoxia cell signaling through RNA posttranscriptional modifications. Using the differentially regulated genes of the hypoxia culture model we have also shown that it is possible to filter genetic models systems such as human chronic/acute kidney disease or rat heterogenous stock proteinuria to narrow down novel mechanisms for genetic influence to hypoxia response. In doing this, we nominated and validated the role of SEPTIN8 in hypoxia response cell integrity, where dysregulation of SEPTIN8 can further propagate complications of hypoxia to disease progression.