Interleukin‐6 Induced Differential Gene Expression in mDCT15 Cells

Abstract

Hypertension (HTN) is a major clinical concern. HTN can be attributed to excessive sodium (Na+) reabsorption by cells in the aldosterone‐sensitive distal nephron (ASDN), resulting in decreased water excretion and a systemic increase in blood pressure. The need for rapid identification of new therapies is important, since anti‐hypertensive drugs are often unsuccessful in regulating blood pressure.Prior research demonstrates an association between inflammation and the progression of HTN. It has been estimated that high intake of Na+ in the ‘Western’ diet along with increased extracellular Na+ may drive a systemic inflammatory response. These responses include increased cytokines, such as interleukin‐6 (IL6). IL6 has been shown to be consistently increased in hypertensive patients. Additionally, our previous data suggests that IL6 can activate the mineralocorticoid receptor (MR) both in vitro and in vivo, as well as increase blood pressure. Our data also suggest a strong mechanism for reactive oxygen species (ROS)‐ and Rac1‐driven activation of the MR, leading to distal Na+ transporter activity. In this study, we utilized RNA sequencing (RNAseq) revealing novel targets driving the IL6‐mediated activation of the MR in the DCT2. We hypothesize that IL6 induces a specific DCT2 transcriptome.To test this hypothesis, we used mDCT15 cells that model the DCT2; an area critical for MR‐mediated Na+ reabsorption. mDCT15 cells were treated with IL6 (100ng/mL, 24 hours) or vehicle (n=4, each), then total RNA isolated. RNA was then converted to mRNA and used to build a library. Data from the Illumina‐sequenced transcriptome underwent comparative analysis to identify significant differentially expressed transcripts between vehicle and IL6 treated cells.Out of 10849 shared transcripts, 64 were specifically expressed with IL6 treatment (Fig. 1). Following Gene Ontology (GO) Enrichment, the gene ratio for ribosomal machinery and cytoskeleton remodeling was significant. We also uncovered several targets of interest which were differentially expressed, as shown by adjusted p value (p‐adj), following IL6 treatment. The genes Gstp1 (5.48×10−10, p‐adj) and HSP90 (3.03×10−6, p‐adj) were both significantly increased following IL6 treatment. Gstp1 encodes for glutathione S‐transferase, which is important in reducing cellular ROS, while Hsp90 is critical for the movement of the MR into the nucleus. Additionally, Arhgef16 (0.0033, p‐adj) was significantly reduced following IL6 treatment; this protein is a Rho‐guanine exchange factor (RhoGEF) and may be important in the regulation of Rac1. These targets are in line with data from our laboratory suggesting that the IL6‐induced increases in ROS generation and Rac1 activation induce MR translocation and activation. Together, these genes represent novel targets for alternate IL6‐mediated MR activation.Support or Funding InformationDK115660, ASN Gottschalk Award to BMW; DK110409 to DCEInterleukin 6 Stimulated Differential Gene Expression in mDCT15 Cells.Figure 1