Abstract 34: Deletion of Nos1 In The Macula Densa Induces Salt-sensitive Hypertension

Abstract

Nitric oxide (NO) released from NO synthase 1 (NOS1) which is highly expressed in the macula densa (MD), blunts tubuloglomerular feedback (TGF) response and dilates the afferent arteriole. However, the role of MD NOS1 and TGF in regulation of chronic salt-water balance and blood pressure is unknown. We developed a strain of tissue-specific NOS1 knockout mice to examine the significance of NOS1 in the MD. We first developed a strain of MD Cre mice using the sodium, potassium, 2 chloride cotransporter (NKCC2) promoter. The NKCC2 promoter was cloned into the plasmid pBS594 to create the pNKCC2-Cre transgene. To characterize this new Cre strain, we crossed the NKCC2-Cre mice with an EGFP reporter strain (Rosa). Cre activity measured by EGFP fluorescence was found in MD and was further confirmed via immunofluorescent staining in isolated perfused MD and found to be limited to the MD and not in the distal tubule, indicating a valid NKCC2-Cre strain. Next, we generated tissue specific NOS1 knockout mice by crossing NKCC2-Cre mice with NOS1 flox/flox mice, which target the exon-6 of NOS1, therefore deletes all splice variants of NOS1. We confirmed NOS1 KO by immunohistochemistry and found no staining of NOS1 in the MD in KO mice, while NOS1 was abundant in the MD of WT mice. To measure NO generation in the MD, we perfused isolated MD and loaded with fluorescent dye DAF-2. When we switch tubular NaCl from 10 to 80 mM, NO generation in the MD was from 114 ± 12 to 165 ± 11 units/min in WT mice, while in KO mice NO were absent at both 10 and 80 mM NaCl. (n=5; p<0.01) To measure TGF response, we measured maximal change of stop flow pressure using micropuncture. In WT mice, TGF was 4.5 ± 0.3 mmHg; while in KO mice, TGF was 8.4 ± 0.7 mmHg. (p<0.05) To determine the effect of MD NOS1 on the blood pressure response to salt intake , we measured mean arterial pressure (MAP) by telemetry in mice fed a low salt diet (0.01% NaCl) for 10 days, followed by a high salt diet (8%NaCl) for 14 days. MAP of KO mice increased by 10.5 ± 2.4; while WT mice increased 3.1± 2.7 mmHg (p <0.01, n=5). These results indicate that NOS1 in the MD and TGF play important roles in the regulation of blood pressure in response to changes in sodium intake.