Endothelin-1 inhibits sodium reabsorption by ET(A) and ET(B) receptors in the mouse cortical collecting duct

Abstract

The collecting duct (CD) is a major renal site for the hormonal regulation of Na homeostasis and is critical for systemic arterial blood pressure control. Our previous studies demonstrated that the endothelin-1 gene (edn1) is an early response gene to the action of aldosterone. Because aldosterone and endothelin-1 (ET-1) have opposing actions on Na reabsorption (JNa) in the kidney, we postulated that stimulation of ET-1 by aldosterone acts as a negative feedback mechanism, acting locally within the CD. Aldosterone is known to increase JNa in the CD, in part, by stimulating the epithelial Na channel (ENaC). In contrast, ET-1 increases Na and water excretion through its binding to receptors in the CD. To date, direct measurement of the quantitative effect of ET-1 on transepithelial JNa in the isolated in vitro microperfused mouse CD has not been determined. We observed that the CD exhibits substantial JNa in male and female mice that is regulated, in part, by a benzamil-sensitive pathway, presumably ENaC. ENaC-mediated JNa is greater in the cortical CD (CCD) than in the outer medullary CD (OMCD); however, benzamil-insensitive JNa is present in the CCD and not in the OMCD. In the presence of ET-1, ENaC-mediated JNa is significantly inhibited. Blockade of either ETA or ETB receptor restored JNa to control rates; however, only ETA receptor blockade restored a benzamil-sensitive component of JNa. We conclude 1) Na reabsorption is mediated by ENaC in the CCD and OMCD and also by an ENaC-independent mechanism in the CCD; and 2) ET-1 inhibits JNa in the CCD through both ETA and ETB receptor-mediated pathways.

Keywords: aldosterone; collecting duct; endothelin-1; kidney; mineralocorticoid; sodium reabsorption.

Fig. 1.

Effect of time on Na reabsorption, J_Na, in the cortical collecting duct (CCD) of male and female mice. J_Na during period 1 is not different from period 2 in male (A) or female (B) mice (male control 72.1 ± 17.9 vs. control 76.8 ± 15.7, P = NS, n = 4; female control 61.4 ± 9.1 vs. control 72.1 ± 6.4, P = NS, n = 3). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 2.

Effect of luminal benzamil (1 μm) on Na reabsorption, J_Na, in the CCD of male (A) and female (B) mice. Benzamil significantly inhibited J_Na in male and female mice (male control 72.6 ± 8.6 vs. benzamil 43.8 ± 8.7, *P < 0.05, n = 6; female control 67.8 ± 17.9 vs. benzamil 41.1 ± 18.0, *P < 0.05, n = 6). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 3.

Effect of luminal hydrochlorothiazide (HCTZ; 100 μM) on benzamil-insensitive Na reabsorption, J_Na, in the CCD of male mice. HCTZ did not significantly inhibit J_Na in male mice (control 42.3 ± 4.8 vs. HCTZ 50.3 ± 4.8, P = NS, n = 4). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 4.

Effect of luminal benzamil (1 μM) on Na reabsorption, J_Na, in the OMCD of male mice. Benzamil significantly inhibited J_Na in the OMCD of male mice (control 33.1 ± 5.5 vs. benzamil 4.8 ± 5.3; *P < 0.05, n = 4). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 5.

Effect of benzamil in the presence of endothelin-1 (ET-1) on Na reabsorption, J_Na, in the CCD of male mice. In the presence of ET-1, benzamil did not significantly inhibit Na reabsorption (ET-1 26.8 ± 7.2 vs. ET-1 + benzamil 12.5 ± 3.2, P = 0.12, n = 6). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 6.

Effect of ET-1 on benzamil-insensitive Na reabsorption, J_Na, in the CCD of male mice. In the presence of benzamil, ET-1 did not significantly inhibit Na reabsorption (benzamil 40.7 ± 6.6 vs. benzamil + ET-1 49.3 ± 8.2, P = NS, n = 3). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 7.

Effect of benzamil in the presence of ET-1 and the ET_A receptor inhibitor BQ-123 (A) or ET_B receptor inhibitor BQ-788 (B) on Na reabsorption in the CCD of male mice. Benzamil significantly inhibited J_Na in the presence of ET-1 plus ET_A receptor inhibitor BQ-123 (ET-1 + BQ123 control 79.8 ± 8.9 vs. benzamil 52.8 ± 8.3, *P < 0.05, n = 6) but did not inhibit J_Na in the presence of ET-1 plus ET_B receptor inhibitor BQ-788 (ET-1 + BQ-788 control 79.2 ± 19.4 vs. benzamil 75.9 ± 13.4, P = NS). Extended line connects the means ± SE for the 2 experimental periods.

Fig. 8.

ET-1 receptor inhibitors block ET-1 regulation of Na reabsorption, J_Na. ET-1 significantly inhibited J_Na (basal control 72.6 ± 8.6 vs. ET-1 control 26.8 ± 7.2, P < 0.05, n = 6). Benzamil significantly inhibited J_Na in the absence of ET-1 (basal control 72.6 ± 8.6 vs. benzamil 43.8 ± 8.7, P < 0.05, n = 6) and in the presence of ET-1 plus the ET_A receptor inhibitor BQ-123 (ET-1 + BQ-123 control 79.8 ± 8.9 vs. benzamil 52.8 ± 8.3, P < 0.05, n = 6). Benzamil did not inhibit J_Na in the presence of ET-1 plus the ET_B receptor inhibitor BQ-788 (ET-1 + BQ-788 control 79.2 ± 19.4 vs. benzamil 75.9 ± 13.4, P = NS). *P < 0.05, compared with control of the same condition. **P < 0.05, compared with basal control.