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

Daria V. Ilatovskaya, Tengis S. Pavlov, Vladislav Levchenko, Alexander Staruschenko

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

The epithelial Na+ channel (ENaC) is a key transporter participating in the fine tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCDc14) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCDc14 cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H2DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCDc14 monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCDc14 monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS 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 used 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-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.

Original languageEnglish (US)
Pages (from-to)C102-C111
JournalAmerican Journal of Physiology - Cell Physiology
Volume304
Issue number1
DOIs
StatePublished - Jan 1 2013
Externally publishedYes

Keywords

  • Growth factors
  • Hydrogen peroxide
  • NADPH oxidase
  • Oxidative stress
  • Rac1

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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