The Effects of the Natriuretic Peptide System on Alveolar Epithelium in Heart Failure

Figures

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  Figure 1. Effect of NPs on AFC. The rate of alveolar fluid clearance was decreased in the ANP- and BNP-treated groups, from 0.485 ± 0.01 mL/h in control rats (n = 4) to 0.2581 ± 0.013 mL/h (n = 4) and 0.1856 ± 0.005 mL/h (n = 4) in ANP- and BNP-treated groups, respectively. **** p < 0.001 represents a significant difference in the NP groups as compared to controls. (one-way ANOVA Kruskal–Wallis test). Bars represent mean ± SEM.

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  Figure 2. Effect of ANP treatment on αENaC and Na+, K+ATPase levels. Representative Western blot analysis of αENaC and Na+, K+ ATPase in whole lung tissue lysate and isolated AECII are shown in (A,B,E,F), respectively. (C) Western blot analysis relative quantification of αENaC in lung tissue (saline-treated group n = 4. ANP-treated group n = 5). (D) Western blot analysis relative quantification of Na+, K+-ATPase in lung tissue (saline-treated group n = 3. ANP-treated group n = 5). (G,H) Representative Western blot analysis relative quantification of αENaC and Na+, K+-ATPase in isolated AECII (normal group n = 2. ANP-treated group n = 4). GAPDH was used as a loading control. Quantification of qPCR analysis of αENaC and Na+, K+-ATPase mRNA in isolated AECII, normalized to GAPDH, is depicted in (I,J), respectively. * p < 0.05 represents significant differences between saline-treated groups vs. ANP-treated groups. Values are means ± SEM. (t-test).

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  Figure 3. Effect of ANP treatment on αENaC ubiquitination. αENaC and Nedd4-2 immunofluorescence staining in lung tissue of ANP-treated rats and their control that were treated with saline. (A) Immunofluorescence detected by CY3-conjugated secondary antibodies (shown in red), magnification ×20 scale bar 10 µm. Representative Western blot analysis Nedd4-2 in whole lung tissue lysate and isolated AECII are shown in (B). (C) Western blot analysis relative quantification of Nedd4-2 in lung tissue and isolated AECII (saline-treated group n = 3, ANP-treated group n = 5). GAPDH was used as a loading control. * p < 0.05 and **** p < 0.001 represent significant differences between saline-treated groups vs. ANP-treated groups. (t-test). Values are means ± SEM.

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  Figure 4. The effect of ANP on AECII ultrastructure. Transmission electron microscopy images of AECII of ANP-treated rats (Panel B) and their controls that were treated with saline (Panel A). The images in Panel A1–3 demonstrate normal morphology of AECII; whereas ANP treatment induced abnormal morphology with mitochondrial destruction and damage (red arrowhead) with matrix swelling and collapsed cristae (B1,2,3). In addition, autophagic vacuoles were observed in ANP-treated cells (B2,3). Lysosomal accumulation was also observed in the treated group (B2). M–mitochondria, Lys–lysosome, Srf–surfactant, Av–autophagic vacuoles. Scale bar A1–2, B1–2: 0.5 μm. Scale bar A3, B3: 0.2 μm.

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  Figure 5. Effect of NPs on AFC rate in both compensated and decompensated rats. The rate of alveolar fluid clearance was significantly decreased in compensated CHF rats treated with ANP (0.3967 ± 0.03180 mL/h, n = 3) as compared to the untreated compensated CHF group (0.621 ± 0.014 mL/h (n = 4) * p = 0.033). In decompensated CHF rats, AFC was drastically declined; however, the administration of ANP restored AFC from −3.107 ± 0.1821 mL/h (n = 3) to 0.3667 ± 0.00393 mL/h (n = 3). **** p = 0.0495. Results are expressed as mean ± SEM (Wilcoxon–Mann–Whitney test).

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  Figure 6. αENaC expression in CHF. Immunofluorescence analysis of αENaC in AECII isolated from sham control (A), compensated CHF (B), and decompensated CHF (C). Cells were stained with antibodies against αENaC, and immunofluorescence was detected by Alexa fluor 488-conjugated secondary antibodies (shown in green). In decompensated HF rats, there was a remarkable upregulation of ENaC as compared to sham-operated rats. Images were obtained by a confocal LSM 880 upright microscope. Magnification ×20. Scale bar = 10 μm.

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  Figure 7. The effect of ANP treatment on levels and expressions of αENaC and Na+, K+-ATPase in compensated CHF and decompensated CHF. Representative Western blot analysis of αENaC and Na+, K+-ATPase in isolated AECII are shown in (A,B,G,H), respectively. (C) Western blot analysis relative quantification of αENaC in isolated AECII (sham-n = 2, compensated-n = 4, compensated treated with ANP–n = 3). (D) Western blot analysis relative quantification of Na+, K+ ATPase in isolated AECII (sham-n = 2, compensated-n = 3, compensated treated with ANP–n = 3). Quantification of qPCR analysis of αENaC mRNA in isolated AECII from compensated CHF and compensated CHF-ANP-treated group (E) and sham-n = 2, compensated-n = 3, and compensated treated with ANP–n = 4) (F) Na+, K+-ATPase mRNA in isolated AECII from compensated CHF and compensated CHF-ANP treated group (sham-n = 3, compensated-n = 4, compensated treated with ANP–n = 5). (I) Western blot analysis relative quantification of αENaC ATPase isolated AECII from the decompensated groups and the decompensated ANP-treated group (sham-n = 2, decompensated-n = 3, decompensated treated with ANP–n = 3). (J) relative quantification of Na+, K+ ATPase levels in…

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  Figure 8. ANP-induced αENaC ubiquitination. Representative Western blot analysis of αENaC, Nedd4-2-HAtag, and Na+, K+-ATPase HEK cells that were co-transfected with Nedd4-2-HA tag and PGC plasmids are shown in (A–C), respectively. (D–F) Western blot analysis relative quantification of αENaC, Nedd4-2-HAtag, and Na+, K+-ATPase HEK co-transfected cells after treatment with ANP. GAPDH was used as a loading control. (G,H) Representative Western blot analysis of AKT and p-AKT in co-transfected cells after ANP treatment, respectively. (I,J) Western blot analysis relative quantification of AKT and p-AKT in co-transfected cells treated with ANP. (K) displays pAKT/AKT ratio in co-transfected cells that were treated with ANP as compared to their controls. Control group n = 3, ANP-treated group n = 6. * p < 0.05, ** p < 0.001 represent significant differences between the control group and the ANP-treated group. Values are means ± SEM (t-test).

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  Figure 9. Graphic illustration depicting a model of how natriuretic peptides, αENaC, Na/K-ATPase, Nedd4-2, and AKT in AEC are linked to modulate AFC. AFC: alveolar fluid clearance; AEC: alveolar epithelial cells; ANP: atrial natriuretic peptides; ENaC Ub: ubiquinated ENaC; p-AKT: phosphorylated AKT.

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  Figure 10. The study design. Flow chart that describes the study design and the three research stages. CT, control; WB, Western blotting; CHF, congestive heart failure; AFC, alveolar fluid clearance; NP, natriuretic peptides.