Dietary salt with nitric oxide deficiency induces nocturnal polyuria in mice via hyperactivation of intrarenal angiotensin II-SPAK-NCC pathway

Figures

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  Fig. 1. The effect of salt loading on diurnal polyuria differs in young and aged mice. a Daily urine volume by salt concentration (NSD, 1% HSD, 2% HSD, 4% HSD). NSD: 0.2% Salt diet, 1% HSD: 1% Salt diet, 2% HSD: 2% Salt diet, 4% HSD: 4% Salt diet (NSD,1% HSD: n = 6 mice per group, 2% HSD, 4% HSD: n = 3 mice per group). Statistical analysis was performed using the Tukey–Kramer method b Experimental protocol. c Mean 4-h urinary volume for three consecutive days in young NSD (black) and young HSD mice (blue) (19 weeks old), as measured via automated voided stain on paper (aVSOP). Daily urine volume, diurnal urine volume, and DPi (diurnal polyuria index = diurnal urine volume/daily urine volume). The active period (night period) was from 8:00 p.m. to 8:00 a.m. and the inactive period (daytime period) was from 8:00 a.m. to 8:00 p.m. (both 12 h). DPi was calculated by averaging over three days (n = 6 mice per group). Statistical analysis was performed using the two-tailed Student’s t-test. d. Mean 4-h urinary volume for three consecutive days in aged NSD (black) and HSD mice (red) (80 weeks old). Daily urine volume, diurnal urine volume, and DPi in aged NSD and HSD mice (80 weeks old) (n = 4…

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  Fig. 2. A ‘two-hit’ mouse model of nocturnal polyuria. a Experimental protocol. b Urinary NOx concentration adjusted by urinary creatinine level in NSD, HSD, l-NAME, and L-NAME + HSD groups (n = 5). Statistical analysis was performed using the Tukey–Kramer method. c Mean 24-h urinary volume for three consecutive days in young NSD (black) and young HSD mice (blue) (19 weeks old), as measured via aVSOP. Daily urine volume, diurnal urine volume, and DPi (Diurnal polyuria index = diurnal urine volume/daily urine volume). The active period (night period) is from 8:00 p.m. to 8:00 a.m. and the inactive period (daytime period) is from 8:00 a.m. to 8:00 p.m. (both are 12 h). DPi was calculated by averaging over 3 days (n = 6 mice per group). Statistical analysis was performed using the two-tailed Student’s t-test. d Mean 24-h urinary volume for three consecutive days in l-NAME (black) and l-NAME + HSD mice (red) (19 weeks old). Daily urine volume, diurnal urine volume, and DPi under l-NAME administration (5 mg/dL in drinking water) (n = 6 mice per group). Statistical analysis was performed using the two-tailed Student’s t-test. e Representative images of H&E histological staining (above) and…

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  Fig. 3. Overactivation of NCC during the active period inhibits sodium excretion under NO suppression. a Urinary sodium excretion per day (left) during active (middle) and inactive (right) periods (NSD, HSD, L-NAME: n = 7, l-NAME + HSD: n = 9). Statistical analysis was performed using the two-way ANOVA or the two-tailed Student’s t-test. b Representative immunoblotting (above) and quantitative analysis (below) of renal phosphorylated NCC (pT53), NCC, ENaCα, ENaCβ, ENaCγ, and ACTN in the active phase for the four groups. Data are expressed as the mean ± SEM. *p < 0.05 (n = 6 mice per group). Statistical analysis was performed using two-way ANOVA. c Representative fluorescent immunostaining of renal phosphorylated NCC (pT53) and total NCC for the four groups using serial sections. Original magnification: ×200. Scale bar, 100 μm. All data are expressed as the mean ± SEM, *p < 0.05.

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  Fig. 4. Nocturnal polyuria is improved by treatment with an NCC inhibitor (HCTZ), but not by ENaC inhibitor treatment (amiloride). a Active period urine volume and inactive period urine volume and DPi in NSD and l-NAME + HSD groups after HCTZ administration (n = 5 mice per group). Statistical analysis was performed using the two-tailed Student’s t-test. b Urinary sodium excretion per day during active (left) and inactive (right) periods (HCTZ-: n = 9, HCTZ+: n = 5 mice per group). Statistical analysis was performed using two-way ANOVA or the two-tailed Student’s t-test. *p < 0.05. c Inactive period urine volume and 24-h urine volume and DPi in NSD and l-NAME + HSD groups after amiloride administration. Data are expressed as the mean ± SEM (amiloride-: n = 6, amiloride+: n = 5). Statistical analysis was performed using the two-tailed Student’s t-test. All data are expressed as the mean ± SEM, *p < 0.05.

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  Fig. 5. Intrarenal angiotensin II activates the SPAK–NCC pathway. a Representative immunoblotting (above) and quantitative analysis (below) of renal phosphorylated SPAK and total SPAK in the active phase in the four groups (n = 6 mice per group). Statistical analysis was performed using two-way ANOVA. b Serum aldosterone levels among the four groups (NSD, HSD, L-NAME + HSD: n = 5, l-NAME: n = 4). Statistical analysis was performed using the Tukey–Kramer method. c Representative immunoblotting (above) and quantitative analysis (below) of renal AGT (angiotensinogen) in the active phase among the four groups. Each point is presented as the mean ± SEM. *p < 0.05 (n = 6 mice per group). Statistical analysis was performed using two-way ANOVA. d Schematic summary illustrating the regulation of sodium and urine via the angiotensin II-SPAK–NCC pathway in the kidney of our novel nocturnal polyuria mouse mode. All data are expressed as the mean ± SEM, *p < 0.05.