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The nitric oxide (NO)-generating enzyme, NO synthase-1β (NOS1β), is essential for sodium (Na + ) homeostasis and blood pressure control. We previously showed that collecting duct principal cell NOS1β is critical for inhibition of the epithelial sodium channel (ENaC) during high Na + intake. Previous studies on freshly isolated cortical collecting ducts (CCD) demonstrated that exogenous NO promotes basolateral potassium (K + ) conductance through basolateral channels, presumably K ir 4.1 (Kcnj10) and K ir 5.1 (Kcnj16). We, therefore, investigated the effects of NOS1β knockout on K ir 4.1/K ir 5.1 channel activity. Indeed, in CHO cells overexpressing NOS1β and K ir 4.1/K ir 5.1, the inhibition of NO signaling decreased channel activity. Male littermate control and principal cell NOS1β knockout mice (CDNOS1KO) on a 7-day, 4% NaCl diet (HSD) were used to detect changes in basolateral K + conductance. We previously demonstrated that CDNOS1KO mice have high circulating aldosterone despite a high-salt diet and appropriately suppressed renin. We observed greater K ir 4.1 cortical abundance and significantly greater K ir 4.1/K ir 5.1 single-channel activity in the principal cells from CDNOS1KO mice. Moreover, blocking aldosterone action with in vivo spironolactone treatment resulted in lower K ir 4.1 abundance and greater plasma K + in the CDNOS1KO mice compared to controls. Lowering K + content in the HSD prevented the high aldosterone and greater plasma Na + of CDNOS1KO mice and normalized K ir 4.1 abundance. We conclude that during chronic HSD, lack of NOS1β leads to increased plasma K + , enhanced circulating aldosterone, and activation of ENaC and K ir 4.1/K ir 5.1 channels. Thus, principal cell NOS1β is required for the regulation of both Na + and K + by the kidney., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The collecting duct (CD) concentrates the urine, thereby maintaining body water volume and plasma osmolality within a normal range. The endocrine hormone arginine vasopressin acts in the CD to increase water permeability via the vasopressin 2 receptor (V2R)-aquaporin (AQP) axis. Recent studies have suggested that autocrine factors may also contribute to the regulation of CD water permeability. Nitric oxide is produced predominantly by nitric oxide synthase 1 (NOS1) in the CD and acts as a diuretic during salt loading. The present study sought to determine whether CD NOS1 regulates diuresis during changes in hydration status. Male and female control and CD NOS1 knockout (CDNOS1KO) mice were hydrated (5% sucrose water), water deprived, or acutely challenged with the V2R agonist desmopressin. In male mice, water deprivation resulted in decreased urine flow and increased plasma osmolality, copeptin concentration, and kidney AQP2 abundance independent of CD NOS1. In female control mice, water deprivation reduced urine flow, increased plasma osmolality and copeptin, but did not significantly change total AQP2; however, there was increased basolateral AQP3 localization. Surprisingly, female CDNOS1KO mice while on the sucrose water presented with symptoms of dehydration. Fibroblast growth factor 21, an endocrine regulator of sweetness preference, was significantly higher in female CDNOS1KO mice, suggesting that this was reducing their drive to drink the sucrose water. With acute desmopressin challenge, female CDNOS1KO mice failed to appropriately concentrate their urine, resulting in higher plasma osmolality than controls. In conclusion, CD NOS1 plays only a minor role in urine-concentrating mechanisms.

Background: During high sodium intake, the renin-angiotensin-aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid-electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin-angiotensin-aldosterone system-epithelial sodium channel axis., Methods and Results: Male control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin-converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin-angiotensin-aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls., Conclusions: These data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin-angiotensin-aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) production in collecting ducts is critical for maintaining fluid-electrolyte balance. Rat collecting ducts express both the full-length NOS1α and its truncated variant NOS1β, while NOS1β predominates in mouse collecting ducts. We reported that dynamin-2 (DNM2), a protein involved in excising vesicles from the plasma membrane, and NOS1α form a protein-protein interaction that promotes NO production in rat collecting ducts. NOS1β was found to be highly expressed in human renal cortical/medullary samples; hence, we tested the hypothesis that DNM2 is a positive regulator of NOS1β-derived NO production. COS7 and mouse inner medullary collecting duct-3 (mIMCD3) cells were transfected with NOS1β and/or DNM2. Coimmunoprecipitation experiments show that NOS1β and DNM2 formed a protein-protein interaction. DNM2 overexpression decreased nitrite production (index of NO) in both COS7 and mIMCD-3 cells by 50-75%. mIMCD-3 cells treated with a panel of dynamin inhibitors or DNM2 siRNA displayed increased nitrite production. To elucidate the physiological significance of IMCD DNM2/NOS1β regulation in vivo, flox control and CDNOS1 knockout mice were placed on a high-salt diet, and freshly isolated IMCDs were treated acutely with a dynamin inhibitor. Dynamin inhibition increased nitrite production by IMCDs from flox mice. This response was blunted (but not abolished) in collecting duct-specific NOS1 knockout mice, suggesting that DNM2 also negatively regulates NOS3 in the mouse IMCD. We conclude that DNM2 is a novel negative regulator of NO production in mouse collecting ducts. We propose that DNM2 acts as a "break" to prevent excess or potentially toxic NO levels under high-salt conditions., (Copyright © 2016 the American Physiological Society.)

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETB receptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis., (Copyright © 2015 the American Physiological Society.)

Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid-electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.

Nitric oxide (NO) and NO synthase 1 (NOS1) maintain sodium and water homeostasis. The NOS1α and NOS1β splice variants are expressed in the rat inner medulla, but only NOS1β is expressed in the mouse. Collecting duct NOS1 is necessary for blood pressure control. We hypothesized that NOS1 splice variant expression and NO production in the inner medullary collecting duct (IMCD) are regulated differently in mice and rats by high dietary sodium. Male C57blk/J6 mice and Sprague-Dawley rats were fed a 0.4% (normal salt; NS), or 4% (high salt; HS) NaCl diet for 2 or 7 days. Mean arterial pressure was not altered by HS, whereas urinary sodium excretion in mice and rats was increased significantly. Urinary excretion of nitrate/nitrite (NO(x)) and IMCD nitrite production were significantly greater in mice compared with rats on the HS diet. Western blotting indicated that only NOS1β and NOS3 were expressed in the mouse IMCD and that expression was unaffected by the HS diet at either time point. In contrast, NOS1α was detected in the IMCD of rats, in addition to NOS1β and NOS3. Feeding of the HS diet for 2 days increased NOS1α and NOS1β expression in the rat IMCD and 7 day feeding of the HS diet further increased NOS1β expression. Expression of NOS3 was unchanged by the HS diet at either time point. In conclusion, IMCD NO production in mice and rats is distinctly regulated under both NS and HS conditions, including expression of NOS1 splice variants., (© 2013 The Authors Clinical and Experimental Pharmacology and Physiology © 2013 Wiley Publishing Asia Pty Ltd.)

Purpose of Review: In recent years, renal collecting duct-specific endothelin-1 (ET1), endothelin A (ETA) and endothelin B (ETB) receptors as well as nitric oxide synthase 1 (NOS1) knockout mice have been developed with subsequent identification for an integral role in regulation of sodium water homeostasis and ultimately blood pressure. The focus of this review is to integrate these models and to propose a scheme for the control of sodium excretion by the collecting duct and the endothelin/ETB/NOS system., Recent Findings: NOS1 splice variants are expressed in the kidney, especially in the collecting duct. Mice express predominantly NOS1β in the medulla, with NOS1α and NOS1β in the cortex, whereas rats express NOS1α and NOS1β in both the cortex and medulla. Novel transcription of collecting duct ET1 mediated by epithelial sodium channels, mitochondrial Na/Ca exchangers and glucocorticoids has been determined. ET1 via the ETB receptor increases nitric oxide production in both rat and mouse collecting ducts, suggesting that NOS1β is linked to ET1-dependent NOS activation in the kidney. As well, genetic deletion of NOS1 splice variants in the collecting duct results in a salt-sensitive hypertensive phenotype in mice, much like the collecting duct ET1 and collecting duct ETB knockout mice., Summary: In the collecting duct, the ET1/nitric oxide pathways are intimately linked, and deletion of collecting duct ET1, ETB receptor or NOS1β results in a salt-sensitive phenotype, which is at least partially dependent on dysregulation of sodium and water reabsorption.

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.

Neuronal NOS (nNOS) is a constitutively expressed enzyme that catalyzes the oxidation of L-arginine and water to L-citrulline and the gas nitric oxide (NO). Nitric oxide is involved in regulation of a variety of processes, including: vascular tone, neurotransmission, and ion balance in mammals and fishes. In this study, we have cloned and characterized a putative NOS homologue from the brain of the euryhaline killifish, Fundulus heteroclitus. Killifish NOS has 75% amino acid identity to human nNOS, and phylogenetic analysis groups the killifish sequence with the mammalian nNOS, suggesting that it is a mammalian orthologue. Relative quantitative reverse transcriptase-PCR demonstrated that killifish nNOS mRNA is highly expressed in the brain and gill followed by the stomach, kidney, opercular epithelium, intestine and heart. Immunohistochemistry localized nNOS to nerve fibers and epithelial cells adjacent to mitochondrion-rich cells (ion transporting cell) in the gill, suggesting that nNOS production of NO may contribute to regulation of vascular tone and/or MRC function in the teleost gill.