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. 2013 Jan 1;304(1):C102-11.
doi: 10.1152/ajpcell.00231.2012. Epub 2012 Nov 7.

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

Daria V Ilatovskaya  1 , Tengis S Pavlov, Vladislav Levchenko, Alexander Staruschenko
Affiliations

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

Daria V Ilatovskaya et al. Am J Physiol Cell Physiol. .

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 (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H(2)DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) 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.

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Figures

Fig. 1.
Fig. 1.
NAPDH subunits expression in cultured mouse cortical collecting duct (mpkCCDc14) principal cells. Western blotting confirmed the expression of the main subunits of the NADPH oxidase complex in the mpkCCDc14 cells. Western blot analysis was repeated 3 times with similar results.
Fig. 2.
Fig. 2.
Acute application of epidermal growth factor (EGF) stimulates reactive oxygen species (ROS) formation in the mpkCCDc14 cells. Shown are the representative fluorescent micrographs of the mpkCCDc14 cells loaded with CM-H2DCF-DA and treated with vehicle, the ROS inducer pyocyanin (200 μM), the NADPH complex inhibitor apocynin (500 μM), and EGF (50 ng/ml) in the absence or presence of apocynin. Scale bar shown is common for all the images. Summary graph displays the total fluorescence intensity measured from the images of the mpkCCDc14 cells (n = 24 in each group) treated with different experimental agents. Background fluorescence level was corrected. *Significant difference between groups.
Fig. 3.
Fig. 3.
Acute application of insulin stimulates ROS formation in the mpkCCDc14 cells. Shown are the representative fluorescent micrographs of the mpkCCDc14 cells loaded with CM-H2DCF-DA and treated with vehicle, the ROS inducer pyocyanin (200 μM), the NADPH complex inhibitor apocynin (500 μM), and insulin (100 nM) in the absence or presence of apocynin. Scale bar shown is common for all the images. Summary graph displays the total fluorescence intensity measured from the images of the mpkCCDc14 cells (n = 25 in each group) treated with different experimental agents. Background fluorescence level was corrected. *Significant difference between groups.
Fig. 4.
Fig. 4.
Acute application of insulin-like growth factor-1 (IGF-1) stimulates ROS formation in the mpkCCDc14 cells. Shown are the representative fluorescent micrographs of the mpkCCDc14 cells loaded with CM-H2DCF-DA and treated with vehicle, the ROS inducer pyocyanin (200 μM), the NADPH complex inhibitor apocynin (500 μM), and IGF-1 (100 ng/ml) in the absence or presence of apocynin. Scale bar shown is common for all the images. Summary graph displays the total fluorescence intensity measured from the images of the mpkCCDc14 cells (n = 20 in each group) treated with different experimental agents. Background fluorescence level was corrected. *Significant difference between groups.
Fig. 5.
Fig. 5.
EGF, insulin, and IGF-1 acutely increase transepithelial currents in mpkCCDc14 cells via NADPH-mediated ROS production. Shown are summary graphs of equivalent transepithelial current (Ieq) in mpkCCDc14 principal cells in response to 10 ng/ml of EGF (A), 20 nM of insulin (B), and 100 ng/ml of IGF-1 (C). EGF, insulin, IGF-1 and vehicle were added basolaterally at time 0, and current was normalized to the starting level. For experiments with apocynin, cells were incubated with inhibitor (500 μM) overnight before the experiment. Amiloride (10 μM; arrow) was added to the apical membrane at the end of experiment. Values are means ± SE of at least 6 observations.
Fig. 6.
Fig. 6.
Effects of the NADPH oxidase (NOX) inhibitor ML171 on EGF- and insulin-stimulated transepithelial currents in mpkCCDc14 cells. Shown are the summary graph of equivalent transepithelial current (Ieq) across the monolayer of the mpkCCDc14 principal cells in response to 10 ng/ml of EGF (A) or 20 nM insulin (B). EGF or insulin and vehicle were added basolaterally alone or together with ML171 (20 μM for EGF and 0.5 μM for insulin, respectively; bilaterally) at time 0; current was normalized to the starting level. Amiloride (10 μM; arrow) was added to the apical membrane at the end of experiment. Values are means ± SE of at least 6 observations.
Fig. 7.
Fig. 7.
ROS production by a xanthine (X)-xanthine oxidase (XO) reaction results in the destruction of actin microfilaments and an increase of transepithelial currents in mpkCCDc14 cells. A: reaction of 250 μM of xanthine and 15 mU of xanthine oxidase elevated ENaC-mediated currents as measured by equivalent transepithelial current (Ieq) in mpkCCDc14 cells. Xanthine or xanthine oxidase alone and together and vehicle were applied bilaterally at time 0 and current was normalized to the starting level. Values are means ± SE of at least 6 observations. B: visualization of the actin cytoskeleton by staining with rhodamine-phalloidin in mpkCCDc14 cells before (left) and after (right) treatment with 250 μM of xanthine and 15 mU of xanthine oxidase for 2 h. Scale bar shown is 20 μm.
Fig. 8.
Fig. 8.
Short hairpin (sh)RNA-mediated silencing of Rac1 does not affect integrity of M-1 cells. A: Western blot from control M-1 cells (scrambled shRNA) or a stable cell line expressing shRNA vs. Rac1 (Rac1 KD). Cell lysates were analyzed using anti-Rac1 antibodies (top). Equal loading was verified by anti-β-actin antibodies (bottom). Shown are representative data from 3 experiments. B: representative staining of the actin cytoskeleton with rhodamine-phalloidin in the control (left) and Rac1 KD (right) M-1 cells. C: electron micrographs of the control and Rac1 KD M-1 cell monolayers grown on a permeable support at ×4,000 and ×1,2000 magnifications (top and bottom, respectively). Scale bars are shown.
Fig. 9.
Fig. 9.
Effects of EGF on transepithelial currents in control and Rac1 KD M-1 cells. Shown is a summary graph of equivalent transepithelial current (Ieq) in M-1 cells in response to 10 ng/ml of EGF. M-1 cells were serum-starved overnight before the experiment. EGF and vehicle were added bilaterally at time 0 and current was normalized to the starting level (A). Transepithelial resistance (Req) changes in response to EGF stimulation in wild-type and Rac1 knockdown cells are also shown (B). Values are means ± SE of at least 6 observations. Amiloride (10 μM; arrow) was added to the apical membrane at the end of the experiment.
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