Intestinal Gastrin/CCKBR (Cholecystokinin B Receptor) Ameliorates Salt-Sensitive Hypertension by Inhibiting Intestinal Na+/H+ Exchanger 3 Activity Through a PKC (Protein Kinase C)-Mediated NHERF1 and NHERF2 Pathway

Abstract

Background: The present study directly tested the crucial role of intestinal gastrin/CCKBR (cholecystokinin B receptor) in the treatment of salt-sensitive hypertension.

Methods: Adult intestine-specific Cckbr-knockout mice (Cckbr^fl/fl villin-Cre) and Dahl salt-sensitive rats were studied on the effect of high salt intake (8% NaCl, 6-7 weeks) on intestinal Na^+/H⁺ exchanger 3 expression, urine sodium concentration, and blood pressure. High-salt diet increased urine sodium concentration and systolic blood pressure to a greater extent in Cckbr^fl/fl villin-Cre mice and Dahl salt-sensitive rats than their respective controls, Cckbr^fl/fl villin mice and SS13^BN rats. We constructed gastrin-SiO₂ microspheres to enable gastrin to stimulate specifically and selectively intestinal CCKBR without its absorption into the circulation.

Results: Gastrin-SiO₂ microspheres treatment prevented the high salt-induced hypertension and increase in urine Na concentration by inhibiting intestinal Na^+/H⁺ exchanger 3 trafficking and activity, increasing stool sodium without inducing diarrhea. Gastrin-mediated inhibition of intestinal Na^+/H⁺ exchanger 3 activity, related to a PKC (protein kinase C)-mediated activation of NHERF1 and NHERF2.

Conclusions: These results support a crucial role of intestinal gastrin/CCKBR in decreasing intestinal sodium absorption and keeping the blood pressure in the normal range. The gastrointestinal administration of gastrin-SiO₂ microspheres is a promising and safe strategy to treat salt-sensitive hypertension without side effects.

Keywords: blood pressure; cholecystokinin; gastrins; intestines; sodium.

Figure 1.

Intestinal CCKBR (cholecystokinin B receptor) is involved in the regulation of sodium balance and blood pressure (BP). A, Divisions of mouse gastrointestinal tract. B, CCKBR protein expression in 3 segments of the intestines in Cckbr^fl/fl (wild type [WT]) mice and Cckbr^fl/fl villin-Cre (knockout [KO]) mice. C, Immunofluorescence of Alexa Fluor 488-labeled CCKBR (green), Alexa Fluor 568-labeled villin (red) and DAPI(blue) in human intestine and C57BL/6J mouse jejunum. D, Systolic BP was measured by telemetry and the data were analyzed by Acqknowledge 5.0 software. E, Urine sodium concentration was measured by flame photometry (*P<0.05 vs Cckbr^fl/f^l WT+high-salt (HS), 1-way ANOVA, Tukey test).

Figure 2.

Gastrin-SiO₂ microspheres attenuates the sodium-induced increase in blood pressure (BP). A, Protocol for gastrin-SiO₂ microspheres generation and intestinal location in vivo; gastrin-SiO₂ microspheres location determined by fluorescence emission tomography. B, Immunofluorescence of stomach and jejunum in the 2 groups of mice (n=3/group, *P<0.05 vs control). C and D, Gastrin-SiO₂ microspheres ameliorated the high salt (HS)-elevated systolic BP and urine Na concentration in Dahl salt-sensitive rats fed normal (N, 0.49% NaCl) or HS (8% NaCl) diets (*P<0.05 vs Dahl salt-sensitive control [normal-salt diet], # vs Dahl salt-sensitive + HS diet, 1-way ANOVA, Tukey test). E, Urine potassium excretion was not different among the groups. FITC indicates fluorescein isothiocyanate.

Figure 3.

Gastrin/CCKBR (cholecystokinin B receptor) decreases Na+/H+ exchanger 3 (NHE3) expression and activity in intestinal brush border membrane. A, Immunofluorescence of Alexa Fluor 488-labeled CCKBR (green), and Alexa Fluor 568-labeled NHE3 (red) in human intestine (scan bar is 200 μm) and mouse jejunum (scan bar is 75 μm). B and C, NHE3 expression in the 3 intestinal segments in knockout (KO) and wild-type (WT) mice fed normal (N) or high (H) salt diet (n=4/group, *P<0.01 vs Cckbr^fl/fl WT, 1-way ANOVA, Holm-Sidak test). D, Immunofluorescence of Alexa Fluor 488-labeled F-actin (green) and Alexa Fluor 568-labeled NHE3 (red) in Dahl salt-sensitive rats (scan bar is 200 μm). E, Immunofluorescence of Alexa Fluor 488-labeled NHE3 (green) in WT and KO mice (scan bar is 100 μm).

Figure 4.

Gastrin inhibits Na+/H+ exchanger 3 (NHE3) expression via NHERF1 and NHERF2-mediated protein trafficking. A, Expressions of NHE3, NHERF1, NHERF2, NHERF3, ezrin, and IRBIT in intestinal brush border membranes of knockout (KO) mice and wild-type (WT) mice fed normal (N) or high (H) salt diet (n=4/group, *P<0.01 vs Cckbr^fl/fl WT, 1-way ANOVA, Holm-Sidak test). B, Expressions of NHE3, NHERF1, NHERF2, NHERF3, ezrin, and IRBIT in the intestinal brush border membrane of Dahl salt-sensitive (DSS) rats. Villin was used as an internal marker of intestinal epithelial cells (*P<0.05 vs DSS control [0.49% NaCl], #P<0.05 vs DSS + high salt [HS, 8% NaCl], 1-way ANOVA, Tukey test). C and D, AAV-NHERF1 and AAV-NHERF2 plasmids impair the effectiveness of the gastrin-SiO₂ microspheres in ameliorating the increase in systolic blood pressure (BP) and impairing the increase in urine Na concentration in DSS rats fed HS diet (*P<0.05 vs others, # vs DSS + others, 1-way ANOVA, Tukey test).

Figure 5.

Gastrin-mediated Na+/H+ exchanger 3 (NHE3) inhibition via a phospholipase C (PLC)/PKC (protein kinase C)-dependent manner. A–C, Protein expressions of NHE3, NHERF1, NHERF2, NHERF3, Ezrin, and IRBIT on membranes of Caco-2 cells treated with 140 mmol/L Na⁺ (control), 240 mmol/L Na⁺ (high salt [HS]), HS incubated cells pretreated with gastrin (10⁻⁸ mol/L, HS + gastrin), U73122 (PLC inhibitor, 5×10⁻⁶ mol/L) + gastrin, or Go6983 (PKC inhibitor, 5×10⁻⁶ mol/L) + gastrin (*P<0.05 vs control, # vs HS, & vs HS + gastrin, 1-way ANOVA, Tukey test). KOH indicates knockout mice on high-salt diet; KON, knockout mice on normal-salt diet; WTH, WT mice on high-salt diet; and WTN, control group of WT mice on normal-salt diet.

Figure 6.

Gastrin-SiO₂ microspheres effectively inhibit Na+/H+ exchanger 3 (NHE3) activity caused by NHERF1 and NHERF2-inducedvsmall intestinal brush border membrane NHE3 trafficking, through a phospholipase C (PLC)/PKC (protein kinase C)-dependent pathway.