Nocturnal polyuria is the most common cause of nocturia, which significantly impairs quality of life, especially in the elderly. The clinical phenotype that characterizes nocturnal polyuria results from the presence of multiple pathologies, including polydipsia, chronic kidney disease, heart failure and aging. This complexity makes it difficult to understand the pathogenesis and hinders the construction of animal models that mimic the clinical presentation. Recently, high salt intake has been shown to increase the risk of nocturnal polyuria, and the impact of salt intake on nocturnal polyuria has received much attention. However, even though salt intake is similar among generations, the frequency of nocturnal polyuria increases with age. Thus, we hypothesized that aging affects the relationship between nocturnal polyuria and salt intake. Based on this hypothesis, we established a new nocturnal polyuria animal model and elucidated the molecular mechanisms.

First, we focused on nitric oxide (NO), which declines with age, and examined whether NO production affects the relationship between salt intake and nocturnal polyuria in humans. Healthy kidney transplant donors (n=27) were included in the study. Salt intake was estimated by the salt excretion in 24-hour urine storage and urinary NOx, a metabolite of NO, was measured by the Greiss method. The nocturnal polyuria index was calculated from the frequency volume chart. Next, to establish animal model of nocturnal polyuria and evaluated the underlying mechanisms, 19-week-old C57BL6/J male mice were divided into four groups. They were given either normal water or L-NAME (an inhibitor of NO synthase) for 2 weeks and either a standard diet (NSD: Normal Salt Diet, containing 0.2% salt) or a high salt diet (HSD: High Salt Diet, containing 1% salt): NSD group, HSD group, L-NAME group, and L-NAME + HSD group (n=6, respectively). We studied the following: 1. Urine production rhythm: aVSOP (automated voided stain on paper) method was used to measure urine volume at each time point and to calculate DPi (Diurnal Polyuria index = volume of urine during the inactive period/day) as an index corresponding to the nocturnal polyuria index in humans. 2. Water intake and food intake: they were measured in a metabolic cage and compared in the four groups. 3. Histopathology of the kidneys.  4. Urinary sodium excretion, a factor affecting urine production, was measured by storing urine in a metabolic cage. 5. The protein levels of NCC (Na+-Cl- cotransporter), SPAK (upstream of NCC) in the distal tubule and ENaC1) (epithelial Na cotransporter) in collecting duct, which play an important role in the regulation of urinary Na excretion, were evaluated by western blot. 6.The activity of the systemic RAS system (renin-angiotensin-aldosterone system) was evaluated with serum aldosterone (n=5, respectively). Furthermore, since the renal tubulointerstitium contains elements necessary for the production of angiotensin II, a factor regulating the SPAK-NCC pathway, and its substrate, angiotensinogen, represents the renal local RAS system activity, the renal local RAS system activity was evaluated by western blot of angiotensinogen 7. Thiazide (NCC inhibitor) and amiloride (ENaC inhibitor) were administered to the NSD and L-NAME+HSD groups to determine the effect on DPi.

There was a strong correlation between nocturnal polyuria index and salt intake in humans with low urinary NOx (n=13) (r=0.645, p=0.017). On the other hand, salt intake was not correlated with nocturnal polyuria index in humans with high urinary NOx (n=14). (r=-0.195, p= 0.49) Comparison of correlation coefficients showed a significant difference (p= 0.027).
1. Salt loading under normal water did not change DPi, while salt loading under L-NAME increased DPi (0.23 vs 0.28, P<0.05) (Figure 1). Daily urine volume did not differ significantly among the four groups.2. There was no significant difference in water and food intake between the four groups. 3. Histopathological images of the kidneys in four groups by HE and Masson-Trichrome staining showed no glomerulosclerosis, tubular atrophy or interstitial fibrosis.4. Salt loading under normal water increased sodium excretion during the active phase (0.11±0.01 vs 0.20±0.01), but salt loading under L-NAME suppressed the increase in sodium excretion(0.10±0.01 vs 0.16±0.01). In the inactive phase, salt loading under normal water did not increase Na excretion, but salt loading under L-NAME significantly increased Na excretion(0.02 ± 0.01 vs 0.06 ± 0.01 ). 5 We found a significant decrease in phosphorylated NCC(0.99±0.03 vs 0.68±0.03, p<0.05) and no significant change in the expression of NCC and ENaC after salt loading under normal water. On the other hand, after salt loading under L-NAME, phosphorylated NCC was not significantly decreased (0.86±0.03 vs 0.78±0.03) and the expression of NCC and ENaC was not significantly changed (Figure 2). 6. Serum aldosterone levels decreased after salt loading both under normal water(34.1 vs 13.3) and L-NAME administration(103.0 vs 29.1). Renal angiotensinogen did not change after salt loading under normal water (1.01 vs 1.09), whereas there was a marked increase in renal angiotensinogen after salt loading under L-NAME (1.44 vs 2.77, p<0.05). 
7. Treatment with the NCC inhibitor thiazide did not improve DPi in the NSD group (0.13±0.01 vs 0.15±0.01), whereas it did in the L-NAME+HSD group (0.28±0.01 vs 0.19±0.01). When the ENaC inhibitor amiloride was administered, the NSD group did not show a DPi improvement (0.12±0.01 vs 0.13±0.01) and the L-NAME+HSD group did not show a DPi improvement (0.28±0.01 vs 0.25±0.01)

In subjects with decreased NO production, excessive salt intake was found to increase the risk of the nocturnal polyuria. On the basis of this clinical result, the combination of NO deficiency and a high-salt diet allowed us to create an animal model of nocturnal polyuria. In addition, we found that the hyperactivation of the local RAS system in the kidney leads to the hyperactivation of the SPAK-NCC pathway in the distal renal tubules, resulting in insufficient urinary sodium excretion during the active period and increased urine volume during the inactive period. Treatment with an NCC inhibitor resulted in an DPi improvement, indicating that NCC activation is the cause of nocturnal polyuria.

We have successfully established a novel animal model of nocturnal polyuria by combining NO deficiency and a high-salt diet. The pathogenesis of nocturnal polyuria is a decrease in urinary Na excretion during the active period due to hyperactivation of the RAS-SPAK-NCC pathway in the kidney. The renal RAS-SPAK-NCC pathway may be a promising therapeutic target for nocturnal polyuria.

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