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

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, <i>EDN1</i>. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the <i>EDN1</i> gene, called <i>EDN1-AS</i>. Because <i>EDN1-AS</i> represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of <i>EDN1-AS</i> expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted <i>EDN1-AS</i> 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 <i>EDN1-AS</i>, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, <i>n</i> = 7, <i>P</i> < 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 (<i>n</i> = 4, <i>P</i> < 0.01) and the KO2 clone exhibited a two-fold increase (<i>n</i> = 4, <i>P</i> < 0.01). These results support a role for <i>EDN1-AS</i> as a novel regulatory mechanism of ET-1 expression and cellular proliferation.