Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91(phox) signaling to epithelial sodium channels

Abstract

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

Keywords: acute respiratory distress syndrome; alveolar microenvironment; chronic obstructive pulmonary disease; lung injury; pulmonary edema.

Figure 1.

Advanced glycation end products (AGEs) and soluble receptor for advanced glycation end-products (sRAGE) from bronchoalveolar lavage (BAL) fluid of smokers and nonsmokers. BAL fluid from smokers exhibited (A) higher concentrations of AGEs and (B) lower concentrations of sRAGE. (C) Simple linear regression showing that higher levels of sRAGE correlated with lower levels of AGEs. n = 16/group; *P < 0.05.

Figure 2.

Predictors of epithelial lining fluid (ELF) volume in smokers. ELF volume from smokers and nonsmokers calculated using (A) urea and (B) protein methods. ^#P < 0.05. (C) Linear regression model depicting a characteristic of BAL fluid correlated with the level of sRAGE on the x axis and ELF volume on the y axis. Elevated levels of sRAGE correlate with greater volume of ELF regardless of smoking status (P = 0.07). (D) Linear regression model depicting characteristics of BAL fluid from smokers with sRAGE concentration on the x axis and ELF volume on the y axis. Greater concentrations of sRAGE correlated with greater ELF volume in smokers (P = 0.027). There was not a significant correlation between sRAGE and ELF volume in nonsmokers (P = 0.863).

Figure 3.

Human AGEs (hAGEs) regulate epithelial sodium channel (ENaC) activity via oxidant signaling in rat primary alveolar type 1 cells accessed in lung tissue slices. (A) Immunohistochemistry of rat lung slices probed for RAGE and cell-specific markers (erythrina crista galli lectin for alveolar type 1 cells, lysotracker red, which binds to surfactant-producing lysosomes in alveolar type 2 cells) demonstrate that RAGE is expressed in alveolar type 1 and type 2 cells. Merged fluorescent signals indicate colocalization of RAGE- and cell-type markers; 4′,6-diamidino-2-phenylindole (DAPI)–labeled nuclei. Western blot from alveolar type 2 cell lysate immunoblotted for RAGE confirms that RAGE protein is expressed in type 2 cells. (B) Continuous cell-attached single-channel recording of a primary alveolar type 1 cell accessed from a lung slice preparation. Arrow represents the closed (c) state, with downward deflections from the arrow representing inward Na⁺ channel openings (−40 mV holding potential [−Vp]). Enlarged portions of the representative recording represent control, hAGE treatment, and 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl (TEMPO), a superoxide dismutase (SOD) mimetic, conditions. (C) Results from seven independent observations shown on dot plot with ENaC activity on the y axis. Challenge with hAGEs increased ENaC number of channels (N) and the open probability (Po) (NP_o) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017), and the addition of TEMPO decreased ENaC NP_o to 0.15 ± 0.07 (P = 0.01). (D) Point amplitude histograms demonstrate a challenge with hAGEs increases highly selective cation (HSC) and nonselective cation (NSC) activity in primary alveolar type 1 cells.

Figure 4.

hAGEs regulate ENaC activity via oxidant signaling in rat primary alveolar type 2 cells. (A) Continuous cell-attached single-channel recording of a primary alveolar type 2 cell accessed from a lung slice preparation. Arrow represents the closed (c) state, with downward deflections from the arrow representing inward Na⁺ channel openings (−40 mV [−Vp] holding potential). Enlarged portions of the representative recording represent control, hAGE treatment, and TEMPO, a SOD mimetic, conditions. (B) Results from eight independent observations shown on dot plot, with ENaC activity on the y axis. Challenge with hAGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025), and the addition of TEMPO decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). (C) Point amplitude histograms demonstrate that a challenge with hAGEs increase HSC channel and NSC channel activity in isolated alveolar type 2 cells.

Figure 5.

Inhibition of RAGE and protein kinase C (PKC) signaling attenuates hAGE-induced activation of ENaC. (A) Results of eight independent observations shown on dot plot with ENaC open probability (P_o) on the y axis. Inhibition of RAGE signaling using FPS-ZM1 attenuated the effect of hAGEs on ENaC activity (ENaC Po: 0.06 ± 0.02 to 0.06 ± 0.02, and then to 0.03 ± 0.01). (B). Results of eight independent observations shown on dot plot with ENaC Po on the y axis. Inhibition of PKC signaling using GF109203X attenuated the effect of hAGEs on ENaC activity (ENaC Po: 0.11 ± 0.03 to 0.09 ± 0.03, and then to 0.11 ± 0.06). NS, nonsignificant.

Figure 6.

Challenge with hAGEs increased gp91^phox–dependent reactive oxygen species (ROS) production in lung tissue slices and isolated primary alveolar type 2 cells. (A) Representative lung tissue slices challenged with hAGEs with and without FPS-ZM1, a RAGE inhibitor, or with and without GF109203X, a PKC inhibitor, and quantification of fluorescence signal shown in the bar graph. hAGEs significantly increased ROS production. Inhibition of RAGE and PKC signaling significantly decreased ROS production compared with samples treated with hAGEs alone. *P < 0.05. (B) Isolated alveolar type 2 cells treated with hAGEs with and without FPS-ZM1 show a significant increase in ROS production as measured by dihydroethidium (DHE) fluorescence. *P < 0.05. (C). SOD2 was ectopically overexpressed in rat isolated alveolar type 2 cells using adeno-associated viral construct, and then the cells were challenged with hAGEs, and ROS production measured using DHE fluorescence. Overexpression of SOD2 significantly attenuated hAGE-induced ROS production.*P < 0.05. (D) Knockdown of gp91^phox significantly attenuated hAGE-induced ROS production in isolated alveolar type 2 cells. gp91^phox small interfering (si) RNA efficiency was assessed by evaluating gene and protein expression; efficiency was approximately 70%. *P < 0.05.

Figure 7.

Challenge with hAGEs increased phosphorylation of p47^phox in isolated primary alveolar type 2 cells. (A) Western blot of immunoprecipitated p47^phox protein that was immunoblotted for p47^phox, showing molecular weight of approximately 47 kD; the slightly larger molecular weight band is IgG. Representative Western blot of immunoprecipitated p47^phox protein immunoblotted for phosphorylated protein and normalized to total protein (lower panel) under control, hAGE, FPS-ZM1 plus hAGE, and GF109203X plus hAGE treatment conditions. Quantification of blot shows that hAGE treatment significantly increased 47^phox phosphorylation, whereas treatment with FPS-ZM1 and GF109203X attenuated the effects of hAGE. (B) Active PKC of isolated primary type 2 cells was determined under control, hAGE, FPS-ZM1 plus hAGE, and GF109203X plus hAGE treatment. hAGE treatment significantly increased PKC activity, an effect that was abrogated with RAGE and PKC inhibition. (C) The left panel is an immunocontrol of immunoprecipitated gp91^phox and cell lysate (both from alveolar type 2 cells) immunoblotted for gp91^phox. The right panel is immunoprecipitated α-ENaC protein that was then immunoblotted for gp91^phox (nicotinamide adenine dinucleotide phosphate [NADPH] oxidases [Nox] 2) under control, hAGE, FPS-ZM1 plus hAGE, and GF109203X plus hAGE treatment conditions. Bar graph of Western blots showing that hAGEs increased coimmunoprecipitation of ENaC-Nox2, an effect that was attenuated when treated with either FPS-ZM1 or GF109203X. *P < 0.05 compared with control, ^#P < 0.05 compared with hAGEs.

Figure 8.

Instillation of hAGEs promotes lung fluid clearance via RAGE–PKC–Nox2 signaling. Line graphs depicting changes in lung fluid clearance in mice receiving a tracheal challenge, with y axis representing lung fluid clearance (I − I_o), where I represents fluid volume at a respective point in time, and I_o is fluid volume at the first X-ray exposure. (A) hAGEs versus FPS-ZM1 plus hAGEs, (B) GF109203X plus hAGEs versus diphenyliodonium (DPI) plus hAGEs, (C) GF109203X versus GF109203X plus hAGEs. More positive values represent greater fluid clearance. n = 10 per group. N.S., nonsignificant. (D) Lung wet-to-dry weight ratios from mouse lung instilled with hAGEs, FPS-ZM1 plus hAGEs, GF109203X plus hAGEs, and DPI plus hAGEs (*P < 0.05; n = 5 mice/group). (E). Lung wet-to-dry weight ratios from C57Bl/6 mice given a tracheal instillation of scramble, RAGE, or gp91^phox siRNA, and then (24 h later) challenged with hAGEs (*P < 0.05; n = 5 mice/group). Western blots of siRNA instilled lung probed for gp91^phox or RAGE; siRNA decreased protein expression roughly 30% (n = 3).