Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention

Abstract

In vitro studies suggest that collecting duct-derived (CD-derived) endothelin-1 (ET-1) can regulate renal Na reabsorption; however, the physiologic role of CD-derived ET-1 is unknown. Consequently, the physiologic effect of selective disruption of the ET-1 gene in the CD of mice was determined. Mice heterozygous for aquaporin2 promoter Cre recombinase and homozygous for loxP-flanked exon 2 of the ET-1 gene (called CD-specific KO of ET-1 [CD ET-1 KO] mice) were generated. These animals had no CD ET-1 mRNA and had reduced urinary ET-1 excretion. CD ET-1 KO mice on a normal Na diet were hypertensive, while body weight, Na excretion, urinary aldosterone excretion, and plasma renin activity were unchanged. CD ET-1 KO mice on a high-Na diet had worsened hypertension, reduced urinary Na excretion, and excessive weight gain, but showed no differences between aldosterone excretion and plasma renin activity. Amiloride or furosemide reduced BP in CD ET-1 KO mice on a normal or high-Na diet and prevented excessive Na retention in salt-loaded CD ET-1 KO mice. These studies indicate that CD-derived ET-1 is an important physiologic regulator of renal Na excretion and systemic BP.

Figure 1

Constructs used in generating CD-specific KO of the ET-1 gene. The top construct, AQP2-Cre, contains 14 kb of the human AQP2 promoter, an amino-terminus nuclear localization sequence (NLS), a carboxy-terminus HSV glycoprotein D epitope tag, Cre recombinase, and an SV40 PolyA sequence. The bottom construct, floxed ET-1 gene, contains loxP sites flanking exon 2 of the ET-1 gene.

Figure 2

In situ hybridization using an antisense probe for ET-1 mRNA in renal inner medulla from mice containing floxed ET-1 (A), CD ET-1 KO (B), and only the AQP2-Cre transgene (D). (C) The results obtained using the sense probe for ET-1 mRNA in floxed mice. Note that the staining is in a CD pattern. Images shown (×400) are representative of five mice in each group.

Figure 3

Colocalization of YFP expression and AQP2 immunofluorescence in mice doubly heterozygous for ROSA26-YFP and AQP2-Cre. A representative photomicrograph is shown from five separate animals. Images are ×600.

Figure 4

Differences in systolic BP (A) and urinary sodium excretion (UNaV) (B) in control and CD ET-1 KO mice on a normal or high-Na diet. n = 9 per data point. *P < 0.01 vs. control; **P < 0.001 vs. control and P < 0.01 vs. CD ET-1 KO normal Na diet.

Figure 5

Systolic BP in control and CD ET-1 KO mice on a normal or high-Na diet with or without amiloride (3 mg/kg/day) or furosemide (4.2 mg/kg/day). n = 5–9 per data point. *P < 0.01 vs. control, **P < 0.001 vs. control and P < 0.01 vs. CD ET-1 KO normal Na; P < 0.05 vs. ET-1 normal Na; ^#P < 0.01 vs. CD ET-1 KO high Na.

Figure 6

Differences in urinary sodium excretion between control and CD ET-1 KO mice on a normal or high-Na diet. n = 6–9 per data point. *P < 0.01 vs. control.