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Kidney Week

Abstract: PO1400

Kidney Osmoregulation Is Regulated by RNA Polymerase Pausing

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Watts, Jason A., Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, United States
  • Soerianto, Winny, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States
  • Burdick, Joshua T., Howard Hughes Medical Institute, Chevy Chase, Maryland, United States
  • Cheung, Vivian G., Howard Hughes Medical Institute, Chevy Chase, Maryland, United States
Background

Osmoregulation is a complex but critical component of renal physiology that relies on the regulation of gene expression. While many genes and some transcription factors that are involved in osmoregulation have been identified, the initiating regulatory step that triggers the gene expression response to changes in osmolarity remains unknown. To address this knowledge gap, we identified the pausing of RNA polymerase II as a key regulatory step.

Methods

We used the Precision nuclear Run-On and Sequencing assay (PRO-seq), to identify locations of nascent RNA bound to actively transcribing RNA polymerases in inner medulla collecting duct cells (IMCD).

Results

We began by studying Akr1b3, the gene that encodes aldose reductase which is the enzyme that reduces glucose to sorbitol and is essential for osmoregulation in the kidney. We exposed IMCD cells to increasing concentrations of sodium chloride and confirmed that the transcript expression of Akr1b3 increased in a dose-dependent manner. Then we asked how Akr1b3 is regulated transcriptionally. The PRO-seq assay was used to trace the first steps in the synthesis of RNA transcripts for aldose reductase. In IMCD cells at baseline, there is an accumulation of RNA Pol II in the promoter of Akr1b3 consistent with RNA polymerase pausing. Under hypertonic conditions, the paused polymerase is released resulting in the synthesis of aldose reductase transcripts, and subsequent increase in Akr1b3 gene expression level. We confirmed this result by treating IMCD cells with flavopiridol, a drug that increases the stability of proteins in the pausing complex, and Akr1b3 was no longer induced in hypertonic conditions. Next by RNA-sequencing, we found that in addition to Akr1b3, there are over 500 osmotically-induced genes that are regulated by RNA polymerase pausing. These include membrane transporter proteins such as the sodium/myo-inositol cotransporter (Slc5a3) as well as protein chaperones like heat shock protein H1 (Hsph1).

Conclusion

These results indicate RNA Pol II pausing plays a key role in the regulation of gene expression during osmoregulation. In this presentation, I will present data from this study and discuss the tight coupling of gene regulation with renal physiology.

Funding

  • Private Foundation Support