Nephron‐wide deletion of NOS3 impairs salt excretion and causes hypertension during high salt intake via altered NKCC2 activity

Abstract

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.