IL‐6 Increases ROS via Rac1, Leading to Mineralocorticoid Receptor and ENaC Activation

Abstract

HTN is an inflammatory disease characterized by excessive sodium (Na+) reabsorption and increased pro‐inflammatory cytokines, such as interleukin 6 (IL‐6). Aldosterone (Aldo) is the primary ligand for the mineralocorticoid receptor (MR), which is expressed in the aldosterone‐sensitive portion of the distal nephron (ASDN). Although studies show benefits of MR inhibition in HTN, Aldo levels are not always increased, implying an alternate MR activation pathway. The epithelial sodium channel (ENaC) is in both the late distal convoluted tubule (DCT2) and cortical collecting duct (CCD), and is a major Na+ transport protein. We have shown that IL‐6 activates the MR in vitro, and that pharmacological Rac inhibition reduces mineralocorticoid response element (MRE) transcription. We have also shown that IL‐6 increases reactive oxygen species (ROS). Therefore, we hypothesized that IL‐6 activates the MR through increases in ROS generation and Rac1 activity, leading to increased ENaC activity.Since our data show that IL‐6 induces nuclear MR translocation, we tested if Rac1 affects IL‐6 mediated MR translocation. We co‐transfected MR‐eGFP tagged constructs and Rac1 expression vectors into murine distal convoluted tubule cells (mDCT15). We found IL‐6 mediated MR translocation decreased after Rac1 inhibition, or inhibition of MR activation; therefore, we investigated the role of Rac1 in IL‐6 mediated ENaC activity in the CCD and DCT2. We measured transepithelial voltage and resistance using a voltohmmeter, and calculated current in both mDCT15 and cortical collecting duct cells (mpkCCD) (n=4–15/group). Cells were transfected with vectors containing wild‐type Rac1 (WT), Rac1 dominant‐negative (DN Rac1) or constitutively active Rac1 (CA) and then treated with vehicle, Aldo [100nM], or IL‐6 [100ng/mL]. Data are shown as a fold change of current before and after treatments (1hr). We observed a significant increase in amiloride‐sensitive current in both mDCT2 and mpkCCD cells following both Aldo and IL‐6 treatments. Interestingly, the current was higher in the DCT2 than in the CCD (10.04 vs.1.98‐fold change/baseline). We also found that DN Rac1 completely inhibited IL‐6‐induced Na+ current (−1.16‐fold change/baseline) and that IL‐6 did not further increase current in cells with Rac1 CA (See Figure 1). Our data are the first to investigate ENaC activity in DCT2. Additionally, we show that IL‐6 is a strong stimulus for ENaC activity in both the DCT2 and CCD.Since IL‐6 increases ROS production in the DCT2, we investigated the role of Rac1 in this mechanism. mDCT15 cells were transfected with Rac1 inhibition and treated with IL6 [100ng/mL]. Cells were stained with dihydroethidium (DHE, ROS stain) and with confocal microscopy, we visualized ROS production. Our preliminary data show that Rac1 knockdown inhibits IL‐6 mediated ROS production (832.5±18 vs. 1073±29 mean DHE fluorescence, p=0.001).Together, our data show that IL‐6 may play an important role in MR activation, coupled with Rac1 and ROS‐generation in the distal nephron. Our data reveal a potential mechanism of MR activation during HTN, without increased Aldo levels.Support or Funding InformationDK115660, ASN Gottschalk Award to BMW; DK110409 to DCERac1 Inhibition Reduced IL‐6‐Mediated ENaC Current in mDCT15 cell line.Data are shown as fold‐changes in current from baseline (n=4–15, *p<0.05 ANOVA, Kruskal‐Wallis).Figure 1