Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1α Knockout and Wild-Type Mice

Abstract

We recently showed that α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1β accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1αKO model. Consequently, we hypothesized that inhibition of NOS1β in NOS1αKO mice induces salt-sensitive hypertension. NOS1αKO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)-treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1αKO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈ 40% after a high-salt diet in both NOS1αKO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈ 15 mm Hg similarly in NOS1αKO and WT mice treated with 7-nitroindazole. We conclude that NOS1β, but not NOS1α, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading.

Keywords: 7-nitroindazole; arterial pressure; hypertension; sodium; telemetry.

Figure 1. Splice variants of NOS1

mRNA of splice variants of NOS1 were measured by real-time PCR in the macula densa isolated with laser capture microdissection (A). Splice variants of NOS1 protein were measured with a C-terminal antibody in renal cortex and compared with that in brain (B). Bands of Western blot were semi-quantified by densitometry (C). * p<0.01 vs NOS1α, # p<0.01 vs C57BL/6

Figure 2. NO generation by the macula densa

NO generation was measured with a NO-sensitive fluorescent dye in isolated perfused juxtaglomerular apparatus. A. NO generation during TGF when tubular NaCl was increase from 10 to 80 mM between NOS1αKO and WT mice. *p<0.05 vs 10 mM NaCl. B. TGF-induced NO generation in NOS1αKO and WT mice between with and without 7-NI treatment. *p<0.01 vs without 7-NI.

Figure 3. Measurement of GFR in conscious mice

GFR in conscious mice was measured with a single injection of FITC-inulin. GFR was compared between NOS1αKO and WT mice fed a normal salt diet (NS), first period high salt diet (HS1), second period high salt diet without 7-NI (HS2) and with 7-NI (Hs(2)+7-NI). * p<0.01 vs NS.

Figure 4. Kidney clearance function measurement

GFR (A), urine flow rate (B) and sodium excretion rate (C) were measured in mice following an acute volume expansion by a bolus infusion of saline of 3% body weight. Kidney clearance function was measured during 0–60 min, and 60–90 min after volume expansion. * p<0.05 vs basal, # p<0.05 vs with 7-NI.

Figure 5. Measurement of MAP in response to a high salt diet with and without 7-NI

MAP was measured with telemetry between NOS1αKO and WT mice fed a normal salt diet (NS), first period high salt diet (HS1), second period high salt diet (HS2) without 7-NI and with 7-NI. A. Changes in MAP over time. * p<0.01 vs basal and w/o 7-NI in C57BL/6 mice, # p<0.01 vs basal and w/o 7-NI in NOS1αKO mice. B. Delta changes in MAP in mice without 7-NI treatment. C. Delta changes in MAP in mice with 7-NI treatment. * p<0.01 vs high salt.