IL6 Mediated Activation of the Mineralocorticoid Receptor via Rac1 and Reactive Oxygen Species (ROS) in Hypertension Public

Abstract

HTN is an inflammatory disease characterized by increased sodium (Na+) reabsorption and pro-inflammatory cytokines, like interleukin 6 (IL6). Although studies show a reduction in blood pressure with mineralocorticoid receptor (MR) antagonists, aldosterone (primary MR ligand) levels are not increased. This implies an alternate MR activation pathway. Herein, we investigate the L-NAME/High-Salt salt-sensitive hypertension (SS-HTN) mouse model. We note a ~2-fold increase in renal IL6 mRNA transcripts and increased expression of two primary Na+ transporters, epithelial sodium channel (ENaC) and sodium-chloride cotransporter (NCC). We have shown that IL6 activates the MR in vitro, and that Rac inhibition reduces mineralocorticoid response element-mediated transcriptional activation. We have also observed that IL6 increases reactive oxygen species (ROS) production, a key factor in HTN. We hypothesized that IL6 activates the MR through Rac1 and ROS generation, increasing ENaC activity. Since our data show that IL6 activates the MR and ROS production, we tested if Rac1 affects IL6-mediated MR nuclear translocation and ROS production in murine distal convoluted tubule cells (mDCT15). IL6-mediated MR translocation decreased after Rac1 knockdown. Given the reduction in MR activation, we next investigated the role of Rac1 in IL6 mediated ENaC activity. We used a voltohmmeter and measured ENaC current in mDCT15 cells. We observed a significant increase in ENaC current following IL6 treatment but noted that transfection of dominant-negative Rac1 inhibited IL6-induced ENaC current. For the role of Rac1 in IL6 mediated ROS production, Rac1 was inhibited in mDCT15 cells and treated with IL6 [100ng/mL]. Cells were stained with dihydroethidium and we visualized ROS production with confocal microscopy. Our data show that Rac1 knockdown inhibits IL6-induced ROS. Since IL6 cannot directly activate Rac1 pathway, we hypothesized that IL6 increases phosphorylation of extracellular signal-regulated kinase (ERK). We treated mDCT15 cells with IL6 and performed a Western Blot analysis. We found IL6 led to a four-fold increase in pERK2 and 0.77 increase in pERK1, while total ERK stayed constant. Together, our data support an alternative mechanism for MR and ENaC activation, in an aldosterone-independent manner. This novel research further adds to the growing literature on the relationship between hypertension and inflammation.

Table of Contents

Introduction and Background………………..……………………………….………..1

Methods……………………….……………………………………………………………..8

Results………………………...…………………………………………………………..…12

Discussion…………………….……………………………………………………………..26

References……………………………………………………………………………………31

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Physiology Biology, Cell
Mot-clé	Renal Sodium Transport Inflammation Hypertension
Committee Chair / Thesis Advisor	Dr. Douglas Eaton, Emory University
Committee Members	Sands, Jeff, Emory University Eisen, Arri, Emory University

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