How Important Is Nitric Oxide Synthesis To The Kidney?

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Part Ⅱ： Nitric oxide signaling in kidney regulation and cardiometabolic health

Mattias Carlström

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Modulation of sodium transport by NO (Nitric oxide)

Sodium and water homeostasis is mainly regulated via the actions of hormones (that is, aldosterone and vasopressin)in the kidney as well as Ang II and endothelin signaling. However, other endogenous compounds that do not circulate at high levels, such as NO (Nitric oxide), contribute substantially to the renal handling of sodium and water via different mechanisms. In general, NO (Nitric oxide) inhibits tubular sodium reabsorption along the nephron; however, the acute and chronic actions of NO (Nitric oxide) in specific tubular segments during health and disease warrant further investigation. In particular, the effects of NO (Nitric oxide) on sodium and fluid reabsorption in proximal tubules are debated as interactions with Ang II and biphasic effects have been reported. These differing effects may be explained by different experimental settings, models and species differences".

Given the short half-life of NO (Nitric oxide) in vivo, its actions are mainly thought to be mediated via autocrine or paracrine signaling. However, NO (Nitric oxide) might also act as an endocrine hormone, potentially via heme-NO (Nitric oxide) signaling. As discussed above, NO in the kidney originates not only from eNOS (Nitric oxide Synthesis) in the vasculature but also from tubular epithelial nNOS (Nitric oxide Synthesis) and potentially iNOS (Nitric oxide Synthesis) during pathological conditions associated with inflammation. Early studies that used pharmacological approaches to investigate the specific role of NOS (Nitric oxide Synthesis)-derived NO (Nitric oxide) on tubular function were sometimes difficult to interpret because non-selective inhibition of systemic NO (Nitric oxide) generation increased blood pressure, reduced renal perfusion, and impacted renal autoregulatory mechanisms. However, subsequent studies using more selective pharmacological inhibitors or genetic knockout approaches demonstrated that NOS (Nitric oxide Synthesis) inhibition can reduce sodium and fluid excretion without inducing substantial hemodynamic changes*5.

Along the nephron, approximately 67% of the filtered sodium load is reabsorbed in the proximal convoluted tubules; 25% in the TAL of the loop of Henle;5% in the distal convoluted tubule, connecting tubule and initial collecting tubule; and 3% in the inner medullary collecting duct4.nNOS (Nitric oxide Synthesis) and/or eNOS (Nitric oxide Synthesis)-derived NO (Nitric oxide) has been reported to inhibit the basolateral sodium-potassium-pump(Nat/K+-ATPase)in the proximal tubule, apical sodium/hydrogen exchanger 3 (NHE3; also known as SLC9A3)in the proximal tubule and TAL of the loop of Henle, apical NKCC2 in the TAL of the loop of Henle and apical epithelial sodium channel(ENaC) in the cortical collecting duct19 (FIG.4). Although nNOS (Nitric oxide Synthesis) is expressed in the distal tubule, its potential role in modulating transporters in this part of the nephron(for example, the Nat/Cl-cotransporter (NCC; also known as SLC12A3))is not clear.

Early studies that used systemic administration of NOS (Nitric oxide Synthesis) inhibitors support an inhibitory effect of NO (Nitric oxide) on proximal tubular sodium reabsorption67. However, a 2014 study using isolated human proximal tubules demonstrated that Ang II dose-dependently stimulated proximal tubular sodium transport(as demonstrated by increased activity of NHE3 and the basolateral Nat'-HCO,-cotransporter)via NO-CGMP-mediated phosphorylation of ERK8. This study also showed that treatment with the NO donor sodium nitroprusside reduced sodium transporter activity in mouse and rat proximal tubules, but had the opposite effect in human proximal tubules. Further investigation is needed to understand the reason for this discrepancy and to clarify if similar phenomena exist for other trans-porters and in other segments of the nephron. The effect of NO on tubular reabsorption could potentially be con-centration dependent and involve interaction with regulatory hormonal systems such as the RAAS. Although the effects of NOon proximal tubular reabsorption are debated, NO clearly has an important role in kidney physiology and compromised NO bioactivity is associated with kidney disease and associated cardiovascular and metabolic disorders7, s,9.

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Tubular handling of NO (Nitric oxide) metabolites

As mentioned above, NO (Nitric oxide) is rapidly metabolized to form nitrite and nitrate, which are mainly excreted by the kidneys. Although urinary excretion of NOx is often reported, this measurement mainly reflects nitrate, which is found in plasma at almost 1,000-fold higher concentrations and has a substantially longer half-life than nitrite(ty~6h versus,~30min); thus, it is much more stable in urine. As diet is a major contributor to the pool of circulating nitrate and nitrite in the body, accumulated excretion of NOx can only be used to estimate NOS (Nitric oxide Synthesis) function during strict dietary restrictions. Moreover, kidney NOS (Nitric oxide Synthesis) activity might substantially influence the total excretion of NOx, at least during high salt intake. Among mice fed a high salt diet, those with deletion of nNOS (Nitric oxide Synthesis) in the collecting duct had approximately 50% lower NOx excretion than controls and developed hypertension 2.

Early studies in young, healthy volunteers showed that only 60% of orally administered 15N-nitrate was excreted in the urine as nitrate within 48h, with minimal amounts(0.1%) excreted in the feces,101. A small amount of 15N labeled nitrate was also excreted as ammonia or urea in the urine(2-4%)and feces (0.2%), but the handling and/or removal of the remaining dose (36-38%)is still not clear. Some nitrate is likely distributed to the muscle pool and elimination from the body via exhalation of nitrogen gas is also possible.

A small clinical study showed that healthy volunteers with normal kidney function (eGFR>60ml/min/1.73m2)had significantly higher fractional excretion of nitrate (median 16.3%;95% CI8.7-22.8)than patients with CKD and eGFR≤30ml/min/1.73 m²（median 10.3%，95% CI96.9-4.4)10.In patients with CKD, renal nitrate clearance correlated positively with kidney function. Reduced fractional excretion of nitrate in patients with reduced eGFR was associated with increased plasma nitrate levels. These findings might be explained by altered glomerular filtration and tubular handling of nitrate during kidney disease, but could also be related to reduced NOS (Nitric oxide Synthesis)-derived bioactivity in patients with CKD, leading to reduced production of oxidized NO (Nitric oxide) metabolites in the circulation to which the kidneys might adapt by reabsorbing more or secreting less nitrate.

A randomized controlled trial that investigated sex differences in renal nitrate handling in adults (n=231)with elevated blood pressure reported that during dietary nitrate restriction, urinary nitrate concentration, amount of nitrate excreted, renal nitrate clearance and fractional excretion of nitrate were significantly lower in women than in men13. However, NO (Nitric oxide) association was observed between plasma nitrate concentration or fractional excretion of nitrate and GFR in either sex. Following high dietary nitrate intake for 5 weeks, fractional excretion of nitrate markedly increased and no sex differences in renal handling of nitrate were observed. This study suggests that tubular nitrate reabsorption might be higher in women than in men, but the underlying mechanisms warrant further investigation.

In the absence of intrarenal generation, the fractional excretion of nitrate correlates linearly with plasma lev-els and has been calculated to be approximately 3-10%in anesthetized dogs and rats, with major reabsorption taking place in the proximal tubuleso4,105. In healthy volunteers, inhibition of carbonic anhydrase using acetazolamide lowered proximal tubular reabsorption of nitrite and nitrate and increased their content in the urine, suggesting a role of carbonic anhydrase-dependent mechanisms in this reabsorption. Evidence suggests that nitrate reabsorption also takes place in later segments of the nephron; clearance and stop-flow studies in dogs showed that inhibition of NKCC2 with furosemide reduced the tubular reabsorption of nitrate from 97% to 87% during inhibition of intrarenal NOS (Nitric oxide Synthesis) and from 90%to 84% without NOS (Nitric oxide Synthesis) inhibition1.

Another possible candidate for nitrate reabsorption is the chloride-bicarbonate exchanger pendrin(also known as SLC26A4), which is expressed in intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct8. In vitro studies have shown that pendrin expression is reduced in mouse cortical collecting ducts and connecting tubules in the presence of NO (Nitric oxide) donors, and upregulated during inhibition of NOs (Nitric oxide Synthesis)1.Sialin (also known as SLC17A5) transports nitrate from the plasma into the salivaryglands4. High apical expression of signaling has been reported in distal tubule cells1, suggesting that this transporter might also contribute to renal reabsorption of nitrate.

Most of the current knowledge regarding the tubular handling of nitrate and nitrite is based on the excretion of nitrate. Both of these anions are freely filtered in the glomeruli but whether similar tubular transport mechanisms exist for nitrate and nitrite along the nephron is unknown. Further studies are needed to identify nitrate and nitrate transporters in the human kidney. Such studies would not only advance understanding of the nitrate-nitrite-NO (Nitric oxide) pathway in health and disease but could potentially lead to novel therapeutic strategies.

The metabolism of NO (Nitric oxide) in kidney

Approaches to restoring NO (Nitric oxide) bioactivity

Despite several decades of research focused on under-standing NO (Nitric oxide) biology and developing novel tools to increase the bioactivity of this signaling molecule in various disorders, particularly in the cardiovascular system, the number of approved clinical applications is limited. Four main approaches could potentially increase NO (Nitric oxide) bioactivity (FIG.5). First, increasing or restoring endogenous NOS (Nitric oxide Synthesis) activity, for example, by supplementation with L-arginine, L-citrulline, or BH; inhibiting arginase activity; lowering endogenous lev-els of NOS (Nitric oxide Synthesis) inhibitors; stimulating hydrogen sulfide (H, S)formation; or using drugs such as statins, ACE2 activators, type-2 angiotensin II receptor(AT,)agonists and Mas receptor agonists that might dampen oxidative stress and facilitate eNOS (Nitric oxide Synthesis) activation. Second, giving substances that directly increase NO (Nitric oxide) generation independently of the NOS (Nitric oxide Synthesis) system, for example, inhaled NO (Nitric oxide) gas or inorganic nitrite, hybrid drugs that attach a NO (Nitric oxide)-releasing moiety to an existing pharmacological agent, increasing H, S signaling, treatment with organic nitrates, or supplementation with inorganic nitrate or nitrite. Third, limiting NO (Nitric oxide) metabolism, for example, by dampening oxidative stress and thereby preventing scavenging of NO (Nitric oxide), and fourth, facilitating downstream signaling pathways, for example, using phosphodiesterase inhibitors,sGC stimulators, or sGC activators. Some of the existing and promising future approaches to increasing NO (Nitric oxide) generation and signaling are discussed below.

Inhaled NO (Nitric oxide) gas

Since the FDA approval of inhaled NO (Nitric oxide) for the treatment of persistent pulmonary hypertension in neonates in 1999, this approach has been used off-label in various clinical settings. Concerns exist regarding chronic use of inhaled NO (Nitric oxide), especially in patients with multiple-organ failure, owing to the risks of methemoglobin formation(due to binding of NO (Nitric oxide) to hemoglobin, which reduces its oxygen-carrying capacity) and development of kidney dysfunction. A systematic review and meta-analysis of randomized trials showed that NO (Nitric oxide) inhalation therapy increased the risk of acute kidney injury (AKI) in patients with acute respiratory distress syndrome(ARDS) but not in non-ARDSpopulations13. The underlying mechanisms likely involve modulation of pre-and post-glomerular arteriolar resistance and altered tubular handling of salt and water, which is supported by previous animal and human studies13. Kidney function and markers of AKI should therefore be closely monitored in patients who require inhaled NO (Nitric oxide) therapy.

Organic nitrates

Nitroglycerin (also known as glyceryl trinitrate) dilates venous capacitance vessels, aorta, medium-to-large coronary arteries and collaterals. This organic nitrate and structurally similar compounds were used to treat angina, acute myocardial infarction, and severe hypertension even before the discovery of the role of NO (Nitric oxide) in physiology4. Chronic use of organic nitrates has been associated with tolerance and risk of adverse effects, including hypotension and endothelial dysfunction, which limit their therapeutic applications.

Arginase inhibition

The NOS (Nitric oxide Synthesis) isoforms compete for L-arginine with two other enzymes, arginase, and arginine methyltransferase, which convert L-arginine into urea and L-ornithine or asymmetric dimethylarginine(ADMA), respectively. ADMA in turn inhibits NOS (Nitric oxide Synthesis) activity by directly competing with L-arginine for binding to NOS (Nitric oxide Synthesis), leading to NOS (Nitric oxide Synthesis) uncouplingl5. Two isozymes of arginase exist; arginase 1 is primarily located in the cytoplasm of hepatocytes and red blood cells16, whereas arginase 2 is located in the mitochondria of several tissues in the body, with high abundance in the kidney(Human Protein Atlas). Increased arginase activity and elevated ADMA levels, together with reduced NO (Nitric oxide) synthesis, have been associated with endothelial dysfunction and increased cardiovascular risk in patients with CKD (Chronic kidney disease). Moreover, arginase inhibition has been shown to improve microvascular endothelial function in patients with coronary artery disease and T2DM9,1x0.

Experimental studies have shown that dietary inorganic nitrate can decrease arginase expression and activity, which may contribute to the salutary effects of nitrate in cardiovascular and metabolic disease21,122. Increased arginase 2 expression and activity have been associated with kidney failure, diabetic kidney disease (DKD), and hypertensive nephropathy, and favorable effects of arginase inhibition have been demonstrated in kid-ney disease models,18. Further studies are required to investigate the potential clinical benefits of attenuating arginase function in patients with kidney disease.

Relationship between chronic kidney disease and Nitric oxide synthesis

H, S formation and signaling

The signaling molecule H, S has many similarities with NO (Nitric oxide) and affects a wide range of physiological functions, including modulation of cardiovascular, renal, and metabolic systems23-12. H, S is formed endogenously in most organs, including the kidney, via enzymatic and non-enzymatic reactions24. Stimulation of H, S production might enhance the NO (Nitric oxide)-sGC-cGMP-PKGpathway by increasing NO production and its downstream signaling. H, S can also increase eNOS (Nitric oxide Synthesis) activation via mechanisms that involve mobilization of intracellular Ca2+ and promotion of phosphorylation26127.In addition, H, S might increase NO (Nitric oxide) production independent of NOS (Nitric oxide Synthesis) via stimulation of XOR-dependent reduction of nitrite to NO128.H, Shas have also been shown to activate sGC and/or directly increase cGMP levels via inhibition of phosphodiesterase. The interactions and crosstalk that occur between the NO and H, S signaling systems are complex and involve the formation ofS/N-hybrid species. Treatment with slow-releasing H, S donors is associated with protective effects in animal models of cardiovascular, kidney, and metabolic diseases2-12s, but these results await further clinical translation.

Phosphodiesterase inhibition

cGMP is hydrolyzed to guanosine monophosphate (GMP)by phosphodiesterase. To date, phosphodiesterase 5(PDE5), which is expressed in various tissues including the cardiovascular and renal systems, has been the main focus of research, but other phosphodiesterase isozymes have also been suggested to modulate NO (Nitric oxide)-mediated cGMP-dependent and independent signaling.

PDE5 inhibitors block cGMP breakdown and thereby lead to increased or prolonged NO (Nitric oxide) signaling. These compounds have been proven to lower blood pressure in preclinical and clinical studies and to exert kidney and cardiovascular protective effects in various experimental models of IRI, heart failure], CKD and DKD12. PDE5 is highly expressed in the kidney (in the glomeruli, mesangial cells, cortical tubules, and inner medullary collecting duct) and the kidney-protective effects of PDE5 inhibitors are thought to extend far beyond their antihypertensive effect3.In 5/6 nephrectomized rats, 8 weeks of treatment with a PDE5 inhibitor initiated immediately after nephrectomy prevented the development of hypertension and ameliorated kidney injury and proteinuria3. However, this profound kidney protection was lost if PDE5 inhibition was initiated at a later stage (that is,4 weeks after nephrectomy)when proteinuria was already evident.

PDE5 inhibitors are currently clinically approved for the treatment of pulmonary hypertension, erectile dysfunction, and lower urinary tract symptoms. However, promising preclinical and early clinical findings suggest that additional therapeutic indications could be possible in the future. For example, a phase II trial demonstrated that once-daily treatment with along-acting PDE5 inhibitor for 12 weeks decreased albuminuria in 256 patients with T2DM and overt nephropathy5. Importantly, this kidney-protective effect was observed despite simultaneous treatment with RAAS blockers and independent of any changes in blood pressure or GFR.

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