Figure 4.

Analysis of urinary Na⁺ excretion, cumulative Na⁺ balance, and renal epithelial sodium channel (ENaC) expression and activity in EP₁^fl/flAqp2Cre⁻ and EP₁^fl/flAqp2Cre⁺ mice on a normal-Na⁺ (NS) or low-Na⁺ (LS) diet. (A) 24-h urinary Na⁺ excretion and cumulative Na⁺ balance in EP₁^fl/flAqp2Cre⁻ and EP₁^fl/flAqp2Cre⁺ mice fed NS or LS diets. N = 10 per group. Data are mean ± SEM. *p<0.05, **p<0.01 vs. EP₁^fl/flAqp2Cre⁻ at the corresponding time period (unpaired Student’s t test) (analysis of the interaction [time × genotype] by repeated-measures ANOVA). (B, C) Immunoblotting and densitometric analysis of ENaC subunits in the renal cortex (B) and medulla (C) in NS and LS treated EP₁^fl/flAqp2Cre⁻ and EP₁^fl/flAqp2Cre⁺ mice. γ-ENaC-probed membrane was stripped and re-probed with anti-β-actin antibody. N = 3–4 per group for statistical analysis. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs. NS; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. EP₁^fl/flAqp2Cre⁻ (ANOVA with the Bonferroni test). (D, E) RT-qPCR analysis of ENaC subunits mRNA in the renal cortex (D) and medulla (E) with GAPDH as an internal control. N = 5 per group. Data are mean ± SEM. **p<0.01, ***p<0.001 vs. EP₁^fl/flAqp2Cre⁻+NS; ^###p<0.001 vs. EP₁^fl/flAqp2Cre⁻+LS (ANOVA with the Bonferroni test). (F) In vivo ENaC activity as reflected by rapid natriuretic responses to amiloride. LS-loaded EP₁^fl/flAqp2Cre⁻ and EP₁^fl/flAqp2Cre⁺ mice were all subjected to a single dose of amiloride (10 mg/kg by intraperitoneal injection) or vehicle treatment, followed by 5-h urine collection, and shown was the change of 5-h urinary Na⁺ excretion, determined by the delta value of 5-h urinary Na⁺ excretion of vehicle and amiloride treatment. N = 11 per group. Data are mean ± SEM. ^***p<0.001 vs. EP₁^fl/flAqp2Cre⁻+LS (unpaired Student’s t test).