ROS production as a common mechanism of ENaC regulation by EGF, insulin and IGF‐1

Abstract

Epithelial Sodium Channel (ENaC) is a key transporter participating in the fine‐tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by EGF, insulin and IGF‐1. It was also proposed that ROS, including H2O2, have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin and IGF‐1 correlate with ROS production in the mpkCCDc14 cells. Western Blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Short circuit current measurements showed an acute increase of ENaC‐mediated current through the mpkCCDc14 monolayer in response to 50 ng/ml EGF, 100 nM insulin or 100 ng/ml IGF‐1. Treatment of mpkCCDc14 cells with EGF, insulin or IGF‐1 also caused an increase in H2O2 production as measured by DCF fluorescence. Pretreatment with NADPH oxidase activity inhibitor apocynin blunted both H2O2 production and increase in ENaC‐mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we created a stable M‐1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride‐senstitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdowned cells. We hypothesize that EGF, insulin and IGF‐1 have a common stimulatory effect on ENaC mediated by ROS production.