Collecting duct-specific knockout of nitric oxide synthase 3 impairs water excretion in a sex-dependent manner

Abstract

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.

Nephron‐Specific Disruption of Nitric Oxide Synthase 3 Causes Hypertension and Impaired Salt Excretion

“…Further, amiloride did not prevent the delayed Na + excretion observed in NOS3 KO mice in response to an acute salt load. In agreement, and despite the finding that a high salt diet increased collecting duct NOS3 activity,22 no effect of collecting duct‐specific NOS3 KO on BP or urinary Na + excretion during normal or high salt intake was observed (ENaC expression was not measured in these studies) 8. In contrast, collecting duct‐specific NOS1 KO caused salt‐sensitive hypertension and Na + retention3 as well as impaired inhibition of ENaC by ET‐1 4.…”

“…We have previously demonstrated that normal kidneys do not have detectable nephron NOS3 immunostaining under baseline conditions or after salt loading 8. Hence, it was not possible to determine the degree of nephron NOS3 protein knockdown beyond the demonstrated reduced whole kidney NOS3 in NOS3 KO mice (compared with control mice) during chronic and acute salt loading.…”

“…Whole kidneys from control and NOS3 KO mice were homogenized, and protein isolation and immunoblotting performed as previously described 8. Antibodies were as follows: NOS3 (1:1000, BD Transduction Labs, Franklin Lakes, NJ), NOS1 (1:500, Santa Cruz, Dallas, TX), NHE3 (1:1000, Millipore, Bedford, MA), phospho (p)‐NHE3 (Santa Cruz), ENaC‐α, ‐β, ‐γ (1:1000, StressMarq, Victoria, BC), p‐NCC (T 53 ) and NCC (1:1000, gift from Dr David Ellison, Oregon Health Sciences University), p‐NKCC2 (T 96/101 ), p‐NKCC2 (S 126 ) and NKCC2 (1:1000/1:20 000, gift from Dr Pablo Ortiz, Henry Ford Hospital), p‐SPAK (S 373 )/p‐OSR1 (S 325 ) (1:1000, Millipore, Temecula, CA), total SPAK and OSR1 (1:1000, gift from Dr Eric Delpire, Vanderbilt University) and mouse monoclonal β‐actin (1:1000, Life Technologies, Carlsbad, CA).…”

“…Knockout of NOS1 specifically in the macula densa7 increased the hypertensive response to a high‐salt diet, while CD‐specific NOS1 knockout (KO) mice had salt‐sensitive hypertension and an impaired natriuretic response 3. In contrast, CD‐specific deletion of NOS3 did not affect urinary salt excretion and blood pressure in response to high salt intake 8. However, other studies, albeit not involving renal cell‐specific gene targeting, have suggested that nephron NOS3 may be an important modulator of renal salt handling and BP.…”

The Impact of the Nitric Oxide (NO)/Soluble Guanylyl Cyclase (sGC) Signaling Cascade on Kidney Health and Disease: A Preclinical Perspective

“…These data were confirmed in vivo in mice lacking nNOS in the collecting duct which exhibited significantly higher sodium retention when kept on a high-salt diet compared to WT-mice [ 49 ]. These natriuretic effects of NO might be mediated also by other NO-synthases and also be sex-specific since it is only in female that collecting duct specific eNOS knockout mice, that the sodium and water secretion was impaired [ 50 ]. In the collecting duct, the effects of NO/cGMP signaling on the regulation of water channel aquaporin-2 (AQP2) need to be described here, although the effects have not been clearly understood.…”