Resveratrol inhibits the epithelial sodium channel via phopshoinositides and AMP-activated protein kinase in kidney collecting duct cells.

Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCDc14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCDc14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P3 reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol's known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4-24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP3, and a sustained late (4-24 h) effect secondary to activation of AMPK

Resveratrol treatment activates AMP-activated protein kinase (AMPK) in mouse cortical collecting duct mpkCCD_c14 cells.

<p>Polarized mpkCCD_c14 cells were fed with media containing resveratrol or vehicle control for the times indicated. Cell extracts (50 µg protein) were separated on a 4–12% SDS gel. (<i>A</i>). Representative immunoblot analysis of the time-dependence of resveratrol-mediated AMPK activation, as measured by immunoblotting of phosphorylated AMPKα (p-AMPKα) in polarized mpkCCD_c14 cells. (<i>B</i>). Density of p-AMPKα bands were quantified and normalized to protein loading using β-actin as control, and means ± SEM were plotted (<i>n</i> = 3), *<i>p</i><0.01 relative to vehicle control at same time point.</p

Resveratrol induces redistribution of PI [3], [4], [5]P₃ reporter GFP-AKT-PH in mpkCCD_c14 cells.

<p>Cultured mpkCCD_c14 cells were transfected with GFP-AKT-PH and examined by live-cell epifluorescence microscopy. Addition of resveratrol (<i>upper panels</i>) caused a rapid redistribution of PI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078019#pone.0078019-Shimkets1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078019#pone.0078019-Hansson1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078019#pone.0078019-Chang1" target="_blank">[5]</a>P₃ from the plasma membrane whereas control-treated cells showed little change (<i>lower panels</i>). Representative data are shown from four independent trials and representative of identical observations.</p

Knockdown of AMPK reduces the late inhibitory effect of resveratrol on amiloride-sensitive currents in mpkCCD_c14 cells.

<p>mpkCCD_c14 cells were lentivirally transduced to express shRNAs directed against AMPKα1 or a control shRNA that does not target any known mammalian gene. (A) Cell extracts (50 µg protein) were separated on a 4-12% SDS gel. Representative immunoblot analysis showing the extent of AMPK knockdown. (<i>B</i>) Densities of AMPK bands were quantified and normalized to protein loading using β-actin as control, and means ± SEM were plotted relative control shRNA. (C) Untransduced (parental), AMPKα1 shRNA or control shRNA transduced mpkCCD_c14 cells were treated with resveratrol starting at time 0. Equivalent short circuit currents (<i>I_sc</i>, µA/cm²) were measured for the times indicated. No significant difference was observed in current values between untransduced cells (no shRNA) and cells transduced to express control shRNA. Values are mean ± SEM of 3 independent experiments with 4 samples per group. *p<0.05, **p<0.01 relative to control shRNA at same time point.</p

Resveratrol inhibits amiloride-sensitive currents in polarized mpkCCD_c14 cells in a dose-dependent.

<p>Polarized mpkCCD_c14 were mounted in modified Ussing chambers and treated with increasing concentrations (25, 50, 75, 100, 150, 200, 300, 400 µM) resveratrol to both apical and basal compartments. Currents were monitored for 30 minutes. 10 µM amiloride was added to the apical compartment at the end of the experiment to determine net Na⁺ transport through ENaC. The amiloride-sensitive % current inhibition was plotted versus concentration (mean ± SEM) and fitted using a sigmoid, 4 parameter curve. The effective IC₅₀ dose is 107 µM. N = 4 for each concentration. Amiloride abolished greater than 95% of the starting current in all conditions tested.</p

Acute inhibition of AMPK with Compound C prevents the late inhibitory effect of resveratrol on amiloride-sensitive currents in polarized mpkCCD_c14 cells.

<p>Polarized mpkCCD_c14 cells were pretreated with media containing 50 µM Compound C or vehicle (DMSO) and incubated for 30 min to allow for AMPK inhibition. In groups pretreated with Compound C, this drug was present throughout the resveratrol treatment. Equivalent short circuit currents (<i>I_sc</i>, µA/cm²) were measured at the times indicated. Values are mean ± SEM of 3 independent experiments with 4 samples per group. *<i>P</i><0.01 relative to cells treated with resveratrol alone at same time point.</p

Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCD(c14) cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK