Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension

Abstract

Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure.

Figure 1. Immunostainings of kidney sections of a Connexin 40 (Cx40)^fl/fl mouse (control) and Tie2-Cre Cx40^fl/fl mouse

(a, c) are immunostained for Cx40 (red) and renin (green). b and d are immunostained for Cx40 (red), renin (green), and CD 31 (blue) as an endothelial cell marker. Arrows indicate endothelial staining, arrowheads renin-producing cells, and asterisks intraglomerular mesangial cells. Note the coexistence of Cx40 and CD31 immunoreactivity in the Cx40^fl/fl kidney (a, b), whereas Cx40 immunoreactivity is absent in the Tie2-Cre Cx40^fl/fl kidney from endothelial cells (c, d). Also, note the similar Cx40 immunoreactivity in the renin-producing cells both in Cx40^fl/fl (a, b) and in Tie2-Cre Cx40^fl/fl (c, d) kidneys.

Figure 2. Immunostainings of kidney sections of a Connexin 40 (Cx40)^fl/fl mouse (control) and renin-Cre Cx40^fl/fl mouse

(a, c) are immunostained for Cx40 (red) and renin (green). b and d are immunostained for Cx40 (red), renin (green), and α-smooth muscle actin (blue) as a smooth muscle cell marker in an afferent arteriole. Arrows indicate endothelial staining, arrowheads renin-producing cells and asterisks intraglomerular mesangial cells. Note the coexistence of Cx40 and renin immunoreactivity in the Cx40^fl/fl kidney (a, b), whereas Cx40 immunoreactivity is absent in the renin-Cre Cx40^fl/fl kidney from renin-producing cells (c, d). Also, note the similar Cx40 immunoreactivity in the endothelium both in Cx40^fl/fl (a, b) and in renin-Cre Cx40^fl/fl (c, d) kidneys.

Figure 3. Blood pressure in awake Connexin 40 (Cx40)^fl/fl, Tie2-Cre Cx40^fl/fl, and renin-Cre Cx40^fl/fl mice as measured by telemetry

Systolic, mean, and diastolic pressures were increased in renin-Cre Cx40^fl/fl mice. Heart rates were not different between genotypes (Cx40^fl/fl, 582±24; Tie2-Cre Cx40^fl/fl, 563±11; renin-Cre Cx40^fl/fl, 622±14 beats/min). Data are means ± s.e.m. of at least nine animals of each genotype. Asterisk indicates P<0.001 vs Cx40^fl/fl and Tie2-Cre Cx40^fl/fl.

Figure 4. Immunostainings of kidney sections of a connexin 40 (Cx40)^fl/fl mouse (control), Tie2-Cre Cx40^fl/fl and renin-Cre Cx40^fl/fl mouse

Immunohistochemistry of renin (green) and α-smooth muscle actin (red) in kidney sections of Connexin 40 (Cx40)^fl/fl (a, d), Tie2-Cre Cx40^fl/fl (b, e) and renin-Cre Cx40^fl/fl (c, f) mice under normal salt diet (a–c) or treated with low-salt diet (0.02%) and enalapril (10 mg/kg) (d–f). Coexpression of renin and α-smooth muscle actin is indicated by yellow color. Dotted lines mark glomeruli. Bar = 50 μm. In Cx40^fl/fl and Tie2-Cre Cx40^fl/fl kidneys, renin-expressing cells are located typically in the walls of afferent arterioles. In renin-Cre Cx40^fl/fl kidneys, renin-expressing cells are located mainly outside the vessel walls.

Figure 5. Plasma renin concentrations in Connexin 40 (Cx40)^fl/fl, Tie2-Cre Cx40^fl/fl, and renin-Cre Cx40^fl/fl mice under normal salt diet or after low-salt diet (0.02%) combined with enalapril (10 mg/kg added to the drinking water) for 7 days

Data are means ± s.e.m., six animals in each group. Asterisks indicate P<0.05 vs normal salt diet.

Figure 6. Renin mRNA abundance in kidneys of Connexin 40 (Cx40)^fl/fl, Tie2-Cre Cx40^fl/fl, and renin-Cre Cx40^fl/fl mice during normal salt diet or after low-salt diet (0.02%) combined with enalapril (10 mg/kg)

Data are means ± s.e.m., six animals in each group. Asterisks indicate P<0.05 vs normal salt diet.

Figure 7. Pressure-dependent renin secretion in isolated perfused kidneys from Connexin 40 (Cx40)^fl/fl, Tie2-Cre Cx40^fl/fl, and renin-Cre Cx40^fl/fl mice

Renin secretion increased with enhanced pressure in renin-Cre Cx40^fl/fl mice, whereas in Tie2-Cre Cx40^fl/fl as well as Cx40^fl/fl controls, the normal inverse relationship was found. Data are means ± s.e.m. of four kidneys of each genotype.

Figure 8. Immunostainings of kidney sections of a renin-Cre Connexin 40 (Cx40)^fl/fl mouse

Higher power magnifications of renin-expressing cells (renin in green) and smooth muscle cells (α-smooth muscle actin in red) in kidneys of renin-Cre Connexin 40 (Cx40)^fl/fl mice under normal salt diet (a) or treated with low salt and enalapril (b). Arrowheads highlight atypically located renin-expressing cells. Note the increase in the number of renin-expressing cells during treatment with low-salt diet, which occasionally extend into the glomerular stalk, and the development of plaque-like patterns. Bar = 50 μm. A, arteriole; G, glomerulus.