BackgroundIn vitro studies suggest that nephron nitric oxide synthase 3 (NOS3) modulates tubule Na+ transport.Methods and ResultsTo assess nephron NOS3 relevance in vivo, knockout (KO) mice with doxycycline‐inducible nephron‐wide deletion of NOS3 were generated. During 1 week of salt loading, KO mice, as compared with controls, had higher arterial pressure and Na+ retention, a tendency towards reduced plasma renin concentration, and unchanged glomerular filtration rate. Chronic high salt‐treated KO mice had modestly decreased total NCC and total SPAK/OSR1 versus controls, however percent phosphorylation of NCC (at T53) and of SPAK/OSR1 was increased. In contrast, total and phosphorylated NKCC2 (at T96/101) were suppressed by 50% each in KO versus control mice after chronic salt intake. In response to an acute salt load, KO mice had delayed urinary Na+ excretion versus controls; this delay was completely abolished by furosemide, partially reduced by hydrochlorothiazide, but not affected by amiloride. After 4 hours of an acute salt load, phosphorylated and total NCC were elevated in KO versus control mice. Acute salt loading did not alter total NKCC2 or SPAK/OSR1 in KO versus control mice but increased the percent phosphorylation of NKCC2 (at T96/101 and S126) and SPAK/OSR1 in KO versus control mice.ConclusionsThese findings indicate that nephron NOS3 is involved in blood pressure regulation and urinary Na+ excretion during high salt intake. Nephron NOS3 appears to regulate NKCC2 and NCC primarily during acute salt loading. These effects of NOS3 may involve SPAK/OSR1 as well as other pathways.

Nitric oxide (NO) inhibits collecting duct (CD) Na and water reabsorption. Mice with CD-specific knockout (KO) of NO synthase 1 (NOS1) have salt-sensitive hypertension. In contrast, the role of NOS3 in CD salt and water reabsorption is unknown. Mice with CD NOS3 KO were generated with loxP-flanked exons 9-12 (encodes the calmodulin binding site) of the NOS3 gene and the aquaporin-2 promoter-Cre transgene. There were no differences between control and CD NOS3 KO mice, irrespective of sex, in food intake, water intake, urine volume, urinary Na or K excretion, plasma renin concentration, blood pressure, or pulse during 7 days of normal (0.3%), high (3.17%), or low (0.03%) Na intake. Blood pressure was similar between genotypes during DOCA-high salt. CD NOS3 KO did not alter urine volume or urine osmolality after water deprivation. In contrast, CD NOS3 KO male, but not female, mice had lower urine volume and higher urine osmolality over the course of 7 days of water loading compared with control mice. Male, but not female, CD NOS3 KO mice had reduced urinary nitrite+nitrate excretion compared with controls after 7 days of water loading. Urine AVP and AVP-stimulated cAMP accumulation in isolated inner medullary CD were similar between genotypes. Western analysis did not reveal a significant effect of CD NOS3 KO on renal aquaporin expression. In summary, these data suggest that CD NOS3 may be involved in the diuretic response to a water load in a sex-specific manner; the mechanism of this effect remains to be determined.

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In vitro studies suggest that nephron nitric oxide synthase (NOS3) regulates urinary Na+ excretion (UNaV) and blood pressure (BP). To assess whether NOS3 is indeed involved in the physiological regulation of UNaV and BP, mice with doxycycline‐inducible nephron‐wide deletion of NOS3 were generated. These mice were homozygous for loxP‐flanked exons 9–12 of the NOS3 gene (contains the calmodulin binding site) and hemizygous for Pax8‐rtTA and LC‐1 transgenes (Pax8 promoter‐rtTA confers nephron‐specific targeting and LC‐1 transgene contains doxycycline/rtTA‐inducible Cre recombinase). Mice were treated with either vehicle (controls) or doxycycline (knockouts, KO) at 1 month of age for 12 days and studied at ~3 months of age. Nephron‐specific NOS3 KO mice had renal‐specific NOS3 gene recombination, reduced NOS3 mRNA levels in microdissected nephron segments, and decreased urine NOx excretion. In response to chronic high salt load, KO mice had increased mean arterial pressure (103 ± 2 mmHg in controls and 118 ± 3 mmHg in KO, N=6, p<0.05), reduced renal NOS3 protein expression (43 ± 8.4% of controls, N=5–6, p<0.05), and enhanced cumulative Na+ retention. In response to acute salt load (1.5 ml saline i.p. injection), KO mice had delayed urinary Na+ excretion vs. controls; this delayed UNaV was abolished by furosemide, but not hydrochlorothiazide or amiloride. After 4h of an acute salt load, KO mice had decreased NOS3 protein expression (19 ± 6.5% of controls, N=5–6, p<0.05) and an increased ratio of phosphorylated/total NKCC2 (162 ± 14.1% of controls, N=5–6, p<0.05). No differences in NHE3, NCC (phosphorylated/total), or ENaC isoforms were noted 4 h after an acute salt load or after chronic salt loading. No sex‐dependent differences in UNaV or BP were observed. In summary, these findings support the notion that nephron NOS3 is involved in acute and chronic control of renal Na+ excretion and BP during high salt intake. Thick ascending limb NKCC2 may be the major transporter involved in nephron NOS3 regulation of renal Na+ excretion and BP.Support or Funding InformationThis research was supported by NIH P01 HL095499This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro studies have suggested that nephron nitric oxide synthase 3 (NOS3) plays a role in the regulation of urinary Na + excretion (UNaV) and potentially contributes to blood pressure (BP) control. To assess whether NOS3 is indeed involved in the physiological regulation of UNaV and BP, mice with doxycycline-inducible nephron-wide deletion of NOS3 were generated. These mice were homozygous for loxP-flanked exons 9-12 of the NOS3 gene (contains the calmodulin binding site) and hemizygous for Pax8-rtTA and LC-1 transgenes (Pax8 promoter-rtTA confers nephron-specific targeting and LC-1 transgene contains doxycycline/rtTA-inducible Cre recombinase). Mice were treated with either vehicle (controls) or doxycycline (knockouts, KO) at 1 month of age for 12 days and studied at ~3 months of age. Nephron-specific NOS3 KO mice had renal-specific NOS3 gene recombination and reduced mRNA levels in microdissected nephron segments. Renal vascular NOS3 mRNA levels were markedly increased in KO mice, presumably as a compensatory response to nephron NOS3 KO. KO mice had modestly increased mean arterial pressure (100 ± 2 mmHg in controls and 103 ± 1 mmHg in KO, N=6, p<0.05) on a normal (0.3% Na + ) salt diet that increased further during high (3.2% Na + ) salt intake (103 ± 2 mmHg in controls and 118 ± 3 mmHg in KO, N=6, p<0.05). KO mice had reduced daily UNaV and greater cumulative Na + retention compared to controls over the 7 days of high salt feeding. PRC tended to be lower in KO mice on a high salt diet compared to controls (85.8 ± 13.5 ng/ml in controls and 60.1 ± 10.3 ng/ml in KO, N=12, p<0.07). Control mice excreted most of an acute I.P. salt load by 4 hr post-load, while KO mice excreted the acute salt load between 5-7 hr after loading. Furosemide, but not hydrochlorothiazide, abolished the differences in UNaV between control and control mice following an acute salt load. No sex-dependent differences in UNaV or BP were observed. In summary, these findings indicate that nephron NOS3 is involved in acute and chronic control of renal Na + excretion and BP during high salt intake. Taken together with previous studies showing that collecting duct-specific KO of NOS3 does not alter UNaV or BP, our data suggest that thick ascending limb is at least partly involved – further mechanistic studies are ongoing.