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Author Notes:

michelle.gumz@medicine.ufl.edu

LD, KS, LJ, CW, BC, KB, and MG conceived and designed research. LD, KS, SM, DB, SB, LJ, KA, RP, and KB performed experiments. LD, KS, SM, DB, and MG analyzed data. LD and MG interpreted results of experiments, prepared figures, and drafted manuscript. LD, SM, DB, SB, LJ, KB, BC, and MG edited and revised manuscript. LD, KS, SM, DB, SB, LJ, KA, RP, CW, KB, BC, and MG approved final version of manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Research Funding:

This work was supported by the National Institutes of Health (NIH) R21AG052861 and 1R01DK109570-01A1 (MG), American Heart Association Grant-in-Aid (16GRNT31220009) (MG), University of Florida Department of Medicine Gatorade Trust (MG), American Heart Association Postdoctoral Fellowship Grants 18POST34030210 (LD), and NIH Grant T32-DK-104721 awarded to the University of Florida (UF) Division of Nephrology (LD).

Keywords:

  • kidney
  • long non-coding RNA
  • proximal tubule cells
  • CRISPR
  • circadian rhythm

EDN1-AS, A Novel Long Non-coding RNA Regulating Endothelin-1 in Human Proximal Tubule Cells

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Journal Title:

Front Physiol

Volume:

Volume 11

Publisher:

, Pages 209-None

Type of Work:

Article | Final Publisher PDF

Abstract:

Endothelin-1 (ET-1) is a peptide hormone that functions as a vasoconstrictor in the vasculature, whereas in the collecting duct of the kidney it exerts blood pressure-lowering effects via natriuretic actions. Aberrant ET-1 signaling is associated with several pathological states including hypertension and chronic kidney disease. ET-1 expression is regulated largely through transcriptional control of the gene that encodes ET-1, EDN1. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the EDN1 gene, called EDN1-AS. Because EDN1-AS represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of EDN1-AS expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted EDN1-AS transcription start site was identified using the ENCODE database and the UCSC genome browser. Two homozygous deletion clones of the element were generated using CRISPR/Cas9. This deletion resulted in a significant increase in the expression of EDN1-AS, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, n = 7, P < 0.05). Phenotypic characterization of these CRISPR clones revealed a difference in cell growth rates. Using a standard growth assay, we determined that the KO1 clone exhibited a three-fold increase in growth over 8 days compared to control cells (n = 4, P < 0.01) and the KO2 clone exhibited a two-fold increase (n = 4, P < 0.01). These results support a role for EDN1-AS as a novel regulatory mechanism of ET-1 expression and cellular proliferation.

Copyright information:

© 2020 Douma, Solocinski, Masten, Barral, Barilovits, Jeffers, Alder, Patel, Wingo, Brown, Cain and Gumz.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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