Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats

Figures

• 

  Figure 1. HS diet elevates blood pressure in a time-dependent manner, reduces endothelium-dependent artery relaxation, and stimulates endothelial ENaC in SS rats. SBP (a), DBP (b), and MAP (c) were measured in SD and SS rats fed NS (0.3%) or HS (5%) diet for 0, 7, 14, and 21 days. ∗represents P < 0.05vs. SS-NS, SS rats fed NS (n = 7 rats for each group). (d, e) Representative traces of ACh-induced artery relaxation in NS or HS diet-fed SD and SS rats and summary of relaxation responses of mesenteric arteries to different doses of ACh under the conditions shown in (d). The first dot in each plot represents the time when Phe (10 μM) was added. The following dots indicate ACh concentrations gradually increased to 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM. ∗indicates P < 0.05vs. SS-NS (n = 6 for each group). (f) Summary of relaxation responses of mesenteric arteries from these rats to different doses of NTG (n = 6 for each group). (g) Representative ENaC single-channel currents generated by split-open artery technique in intact endothelial cells from NS or HS diet-fed SD and SS rats, under the indicated conditions. (h) Summary of calculated ENaC PO obtained from single-channel…

• 

  Figure 2. HS diet increases BMP4 levels both in the serum and in the endothelial cells of SS rats. (a) Serum BMP4 levels were measured at day 21 in NS or HS diet-fed SD and SS rats. ∗represents P < 0.05vs. SS-NS (n = 13 for each group). (b) Representative Western blot and summarized data showing BMP4 protein levels under each condition in endothelial cells isolated from SD and SS rats fed NS or HS diets. ∗represents P < 0.05vs. SS-NS (n = 10 for each group).

• 

  Figure 3. HS diet impairs EDR and stimulates ENaC via a pathway associated with BMP4 in SS rats. (a) Representative traces of ACh-induced artery relaxation in NS or HS diet-fed SS rats; the vascular rings were, respectively, treated with BMP4 (50 ng/mL) and noggin (200 ng/mL) for 4 hours and benzamil (1 μM) for 1 hour before experiments. The first dot in each plot represents the time when Phe (10 μM) was added. The following dots indicate ACh concentrations gradually increased to 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM. (b) Summary of relaxation responses of mesenteric arteries to different doses of ACh under the conditions shown in (a). ∗indicates P < 0.05vs. NS; #indicates P < 0.05vs. HS; &represents P < 0.05vs. NS + BMP4 (n = 6 for each group). (c) Summary of relaxation responses of mesenteric arteries from these rats to different doses of NTG (n = 6 for each group). (d) Representative ENaC single-channel currents in intact endothelial cells attached to a mesenteric artery isolated from NS or HS diet-fed SS rats; mesenteric arteries, respectively, treated with BMP4 (50 ng/mL) and noggin (200 ng/mL) for 4 hours and application of 1 μM benzamil prior to recording. (e) Summary…

• 

  Figure 4. HS diet impairs endothelium-dependent relaxation and stimulates ENaC via p38 MAPK in SS rats. (a, b) Upper panels are representative Western blots and summarized data showing p38 MAPK and p-p38 MAPK protein levels under each condition in endothelial cells isolated from SS rats fed NS or HS diet. ∗P < 0.05vs. NS (n = 6 for each group). (c) Representative traces of ACh-induced artery relaxation in NS or HS diet-fed SS rats, in absence of or in the presence of 20 μM SB202190 for 4 hours. The first dot in each plot represents the time when Phe (10 μM) was added. The following dots indicate ACh concentrations gradually increased to 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM. (d, e) Summaries of relaxation responses of mesenteric arteries to different doses of ACh (d) and to different doses of NTG (e). ∗P < 0.05vs. NS; #P < 0.05vs. HS (n = 6 for each group). (f) Representative ENaC single-channel currents generated from the intact endothelial cells of mesenteric artery isolated from NS or HS diet-fed SS rats, in the absence of or in the presence of 20 μM SB202190 for 4 hours. (g) Summary of ENaC PO obtained from single-channel recordings from the SS rats, under indicated…

• 

  Figure 5. HS activates Sgk1/Nedd4-2 via BMP4 and p38 MAPK signaling. (a) Representative Western blots and summarized data showing BMP4 protein levels in endothelial cells isolated from SS rats either under control conditions or treated with an additional 40 mM NaCl in the culture medium for 24 hours. The data are the means ± SEMs of 6 experiments in each group. ∗indicates P < 0.05vs. control. (b) Representative Western blots and summarized data showing p38 MAPK and p-p38 MAPK protein levels under control conditions or treated with HS additional 40 mM NaCl in the culture medium for 24 hours, in the absence of and in the presence of 100 ng/mL noggin. ∗indicates P < 0.05vs. control; #represents P < 0.05vs. HS (n = 6 for each group). (c–d) Representative Western blots and summarized data showing Sgk1, p-Sgk1, Nedd4-2, and p-Nedd4-2 protein levels under control conditions or treated with additional 40 mM NaCl in the culture medium for 24 hours, in the absence of and in the presence of either 100 ng/mL noggin or 10 μM SB202190 for 24 hours. ∗indicates P < 0.05vs. control; #represents P < 0.05vs. HS (n = 6 for each group). (e–h) Representative Western blots and summarized data showing BMP4, p-p38…

• 

  Figure 6. BMP4 stimulates ENaC by activating p38 MAPK and Sgk1/Nedd4-2. The experiments were conducted in the primary cultured endothelial cells (ECs) isolated from SS rats and the ECs were, respectively, transfected by p38 MAPK control LV (MOI = 10) and p38 MAPK shRNA LV (MOI = 10) to knockdown p38 MAPK. (a, b) Representative Western blots and summarized data showing p38 MAPK and p-p38 MAPK protein levels in primary cultured endothelial cells isolated from SS rats either under control conditions or treated with p38 MAPK control LV or p38 MAPK shRNA LV. ∗P < 0.05vs. control (n = 6 for each group). (c) Summarized data showing p38 MAPK mRNA levels in primary cultured endothelial cells isolated from SS rats either under control conditions or treated with p38 MAPK control LV or p38 MAPK shRNA LV. ∗P < 0.05vs. control (n = 6 for each group). (d–i) Representative Western blots and summarized data showing p38 MAPK, p-p38 MAPK, Sgk1, p-Sgk1, Nedd4-2, and p-Nedd4-2 protein levels in ECs or in p38 MAPK gene manipulated ECs under control conditions or treated with 20 ng/mL BMP4 for 24 hours. ∗indicates P < 0.05vs. control; #represents P < 0.05vs. BMP4 (n = 6 for each group). (j) Representative ENaC…