What Are The Beneficial Effects Of Nitric Oxide On Kidney

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Part Ⅲ： Nitric oxide signalling in kidney regulation and cardiometabolic health

Mattias Carlström

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Abstract

The prevalence of cardiovascular and metabolic disease coupled with kidney dysfunction is increasing worldwide. This triad of disorders is associated with considerable morbidity and mortality as well as a substantial economic burden. Further understanding of the underlying pathophysiological mechanisms is important to develop novel preventive or therapeutic approaches. Among the proposed mechanisms, compromised nitric oxide (NO) bioactivity associated with oxidative stress is considered to be important. NO (nitric oxide) is a short-lived diatomic signalling molecule that exerts numerous effects on the kidneys, heart and vasculature as well as on peripheral metabolically active organs. The enzymatic L-arginine-dependent NO (nitric oxide) synthase (NOS)pathway is classically viewed as the main source of endogenous NO (nitric oxide) formation. However, the function of the NOS (nitric oxide synthase) system is often compromised in various pathologies including kidney, cardiovascular and metabolic diseases. An alternative pathway, the nitrate-nitrite-NO (nitric oxide) pathway, enables endogenous or dietary-derived inorganic nitrate and nitrite to be recycled via serial reduction to form bioactive nitrogen species, including NO (nitric oxide), independent of the NOS (nitric oxide synthase) system. Signalling via these nitrogen species is linked with cGMP-dependent and independent mechanisms. Novel approaches to restoring NO (nitric oxide) homeostasis during NOS (nitric oxide synthase) deficiency and oxidative stress have potential therapeutic applications in the kidney, cardiovascular and metabolic disorders.

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Cardiovascular effects.

Considerable research efforts have focused on the cardiovascular effects of inorganic nitrate supplementation, including the effects on blood pressure, endothelial function, and arterial stiffness. In 2006, the first study to report a blood pressure-lowering effect of nitrate supplementation in healthy adults showed that 0.1 mmol/kg per day of sodium nitrate reduced diastolic blood pressure by a mean of almost 4mmHg (REF.15). A subsequent study that used an approximately threefold higher dose of dietary nitrate, in the form of beetroot juice, demonstrated a more pronounced blood pressure-lowering effect(ie. mean reductions in systolic blood pressure of 10.4mmHg and diastolic blood pressure of 8mmHg), together with vasoprotective and antiplatelet properties of nitrate. Several research groups have since confirmed the blood pressure-lowering effect of nitrate in healthy individuals. In two meta-analyses of these studies, systolic blood pressure was reduced by means of 4.1-4.8mmHg and diastolic blood pressure was reduced by means of 1.7-2.0 mmHg (REFsi5,15). One of these meta-analyses also analyzed the effect of nitrate intake on other cardiovascular risk factors and reported improved endothelial function, reduced arterial stiffness, and reduced platelet aggregation with this intervention15

Numerous experimental studies using various cardio-vascular disease models, which are often associated with kidney and metabolic dysfunction, have also demonstrated favourable cardiovascular effects following treatment with inorganic nitrate, including antihypertensive effects and improved endothelial function8.1s157. The underlying mechanisms that contribute to such effects involve various organ systems and modulation of the RAAS, inhibition of arginase, restoration of eNOS (nitric oxide synthase) function, dampening of sympathetic hyperactivity, and anti-inflammatory and anti-oxidative effects.

A study in 15 patients with hypertension demonstrated that acute dietary nitrate intake improved endothelial function and significantly lowered systolic and diastolic blood pressure. The maximum blood pressure-lowering effect was observed approximately 3-4h after nitrate ingestion when plasma nitrite levels peaked, and the effect lasted for as long as 24h. The first two studies investigating the cardiovascular effects of chronic nitrate supplementation in patients with hypertension were conducted independently of each other in 2015 and generated conflicting results59160. One study showed NO (nitric oxide) significant reduction in blood pressure following 1 week of nitrate supplementation, whereas the other showed a sustained blood pressure reduction following daily dietary intake of nitrate compared with placebo during a 4-week period, without any signs of tachyphylaxisi0. A 2020 study showed that daily intake of nitrate for 5weeks, in the form of either leafy green vegetables or a nitrate pill, did not significantly lower blood pressure in adults with pre-hypertension or stage 1 hypertension compared with intake of alow-nitrate control dietI6. These differing findings are unlikely to be due to differences in the daily dose of nitrate, which was similar in all three studies (approximately0.1 mmol/kg/day), and can not be explained by patient age, body mass index or gender. However, differences in the number of simultaneous antihypertensive drugs, patient demographics, nitrate intake in the placebo group and blood pressure at the time of initiation of nitrate supplementation might be contributing factors. Another clinical trial showed that once-daily nitrate supplementation(approximately 0.1 mmol/kg/day)for 6 weeks improved vascular function in patients with hypercholesterolaemia, which was associated with a mild reduction in blood pressurel62. Potential effects of nitrate supplementation on metabolic and/or kidney functions were not reported in these clinical studies.

Arterial stiffness is associated with aging and is a major risk factor for cardiovascular events such as myocardial infarction and stroke. A systematic review and meta-analysis of randomized controlled trials were conducted to estimate the effects of repeated nitrate administration (at least 3 days)on peripheral and central blood pressure and arterial stiffness in healthy individuals and in patients at increased risk of cardiovascular disease owing to obesity, hypertension, peripheral artery disease, hypercholesterolaemia and/or heart failures. Pooled data from 45 studies using approximately 500mg nitrate per day showed significant reductions in systolic(mean-2.91mmHg)and diastolic blood pressure (mean-1.45mmHg). Analysis of data from three trials that measured central (aortic)blood pressure also showed significant reductions with nitrate supplementation (mean systolic-1.6 mmHg, mean diastolic -2.0mmHg). However, the meta-analysis found NO (nitric oxide) significant differences in the effects of nitrate supplementation on blood pressure between subgroups of patients with different health statuses. Notably, the reductions in blood pressure with nitrate supplementation in this meta-analysis, and in meta-analyses of data from healthy individuals5415, are comparable with those observed in trials of reduced sodium intake and of a Dietary Approaches to Stop Hypertension (DASH) diets,1s. Analysis of data from seven trials that measured augmentation index and pulse wave velocity showed NO (nitric oxide) significant effects of nitrate administration on arterial stiffness83. However, the researchers note that the number of available trials in individuals with additional cardiovascular disease risk factors(i.e. hypertension, diabetes or hyperlipidaemia) was relatively small and their analysis was likely underpowered to detect significant differences. More studies are required to draw reliable conclusions in these patient groups.

Overall, the current evidence for a long-term favour-able cardiovascular effect of nitrate supplementation in patients with cardiovascular disease, including hypertension, is inconclusive. Hence, additional large clinical trials with different doses of nitrate would be desirable.

Cardiovascular effects of nitrate supplementation on kidney function

Metabolic effects.

Impaired metabolic control with obesity and hyperglycaemia is closely coupled with increased risk of DKD, which involves complex glomerular and tubular mechanisms6617. In addition to the well-documented therapeutic benefits of ACE inhibitors and Ang IIrecep-tor blockers in patients with kidney disease 6819, large clinical trials have shown that treatment with sodium/glucose co-transporter-2(SGLT2) inhibitors can reduce albuminuria, risk of CKD (chronic kidney disease) progression and cardiovascular events in patients with T2DM and kidney disease 7o. The favourable effects of SGLT2inhibition are unlikely to be solely mediated by improved glycaemic control. Experimental evidence suggests that they are likely the result of various glomerulotubular mechanisms,17 such as modulation of the myogenic response and TGF as well as tubular reabsorption and potential modulation of renal sympathetic nerve activity. These mechanisms could potentially also indirectly affect NO (nitric oxide) bioactivity.

Mice that lack eNOS (nitric oxide synthase) develop hypertension7 and features that resemble metabolic syndrome(ie. hypertension, dyslipidemia, insulin resistance and obesity)17t. In addition, eNOS (nitric oxide synthase) deficiency in rodents is associated with kidney injuryl75-17 and accelerated progression of CKD (chronic kidney disease)i7817. Almost a decade ago, supplementation with dietary doses of nitrate was demonstrated to reverse features of metabolic syndrome in mice that lacked eNOS (nitric oxide synthase)15. Numerous experimental studies have since confirmed that nitrate supplementation has favourable metabolic effects, which involve modulation of mitochondrial function and oxidative stress, activation of AMP-activated protein kinase(AMPK)signalling and modulation of downstream targets including sterol regulatory element-binding protein 1,acetyl-CoA carboxy-lase, medium-chain specific acyl-CoA dehydrogenase, mitochondrial and peroxisome proliferator-activated receptor-y coactivator la7,181-184. A link between nitrate and/or nitrite supplementation and AMPK activation has also been demonstrated in experimental models of heart failure with preserved ejection fraction1 and IRI of the heart18 as well as in studies of the potential beneficial effects of this supplementation on longevity6. Knowledge of the specific mechanism(s)of AMPK activation in different cell types(for example, hepatocytes, adipocytes, skeletal muscle cells and cardiomyocytes)is limited, but studies have suggested the involvement of nitrate and/or nitrite-mediated modulation of energy-sensing pathways, including inhibition of the target of rapamycin6, activation of sirtuin 3 (REF.18) and PKA, and modulation of mitochondrial-derived ROs7,185.

Experimental evidence suggests that supplementation with nitrate might be a novel, safe and inexpensive therapeutic approach for patients with T2DM at an increased risk of developing DKD8181,187.To date, few clinical trials have tested the potential beneficial effects of nitrate in patients with T2DM. A small trial in 27 patients with T2DM showed no significant effect of 2weeks of nitrate supplementation(250ml beetroot juice daily) on cardio-metabolic functions(that is, blood pressure, endothelial function and insulin sensitivity)8. Another study that investigated the long-term metabolic effects of low-dose nitrate supplementation (250mg per day for 24 weeks)in patients with T2DM found no significant difference in glycaemic control between the nitrate(n=35) and placebo groups(n=29)89. The reason for this lack of effect in these two studies, which contrasts with substantial experimental evidence, might be the fact that almost all the participants were receiving metformin treatment, which is known to activate AMPK10.In a mouse model of cardiometabolic disease,no additional beneficial effects on cardiovascular and metabolic parameters were observed when dietary nitrate supplementation was given in combination with metformin, suggesting similar mechanisms of action. A phase II study that investigated the cardiometabolic effects of nitrite therapy (40 mg, three times daily) for 12 weeks in adults with stage 1-2 hypertension, metabolic syndrome and normal kidney function who were not receiving any medications that affect glucose metabolism showed that nitrite gradually lowered blood pressure during the first 8 weeks of treatment(by approximately-10 mmHg), but blood pressure levels started to return to baseline after 10-12 weeks. Hyperinsulinaemic-euglycaemic clamp studies suggested that nitrite supplementation resulted in a trend towards decreased endogenous glucose production and improved insulin sensitivity. Strikingly, a significant improvement in carotid intima-media thickness and brachial artery endothelial function was observed after 12 weeks of nitrite therapy.

Metabolic effects of NO (nitric oxide) on kidney function

Kidney effects.

Patients with CKD (chronic kidney disease) and those with kidney failure have compromised NOS (nitric oxide synthase) function, reduced NO (nitric oxide) bioactivity 819 and increased cardiovascular morbidity and mortality. Moreover, a positive association between renal nitrate clearance and kidney function has been observed in patients with CKD (chronic kidney disease)0. Studies in adult and paediatric patients with kidney failure have shown that peritoneal dialysis and haemodialysis sessions are associated with disturbed NO (nitric oxide) homeostasis, measured as a reduction in the circulating levels of nitrate, nitrite and cGMP(a marker of NO (nitric oxide) signalling)194-17. Clinical studies are needed to investigate the therapeutic value of restoring NO (nitric oxide) homeostasis, using nitrate and/or nitrite supplementation, in these vulnerable high-risk patients.

In numerous experimental studies, chronic treatment with inorganic nitrate and nitrite has been associated with therapeutic effects such as attenuation of kidney injury and preservation of kidney blood flow and GFR in models of kidney disease with or without coexistent hypertension and metabolic disease8.1s, including models with chronic pharmacological inhibition of NOS (nitric oxide synthase), unilateral nephrectomy combined with a high-salt diet18, two-kidney one clip, deoxycorticosterone acetate salt, Ang II infusion920, ageing and kidney IRI[0203. Based on these studies, several mechanisms have been proposed to contribute to the favourable effects of nitrate and nitrite supplementation. These include dampening of oxidative stress via a reduction in NADPH oxidase activity, increased antioxidant capacity of superoxide dismutase, increased NO (nitric oxide) bioactivity, a reduction in Ang II sensitivity and type I angiotensin II receptor expression in the renovascular system, dampen-ing of renal sympathetic nerve activity and modulation of immune cell phenotypes and mitochondrial function8.

In healthy adults, acute intravenous infusion of nitrite dose-dependently(0.58-5.21 mmol/kg/h) reduced blood pressure compared with placebo but had no significant effect on GFR measured using 5Cr-EDTA clearance20,205. This blood pressure response was augmented in hypertensive compared with normotensive individuals(mean decreases in systolic blood pressure of 17 mmHg vs 10mmHg). The researchers showed that the reduction in blood pressure was associated with a decrease in urinary levels of ENaCy and aqua-porin 2, but the effects of nitrite infusion on fractional sodium excretion were inconsistent(that is, unchanged, decreased or increased)20-20. In healthy individuals, the nitrite-mediated effects were not associated with changes in plasma or urine cGMP levels and were not significantly affected by simultaneous inhibition of XOR, ACE or carbonic anhydrase0. Moreover, dietary nitrate supplementation (approximately 0.1 mmol/kg/day)for 1 week did not significantly change eGFR (measured using creatinine clearance) compared with placebo in healthy young men20.

A systematic review and meta-analysis that investigated various modifiable lifestyle factors showed that higher vegetable intake significantly reduced the risk of CKD (chronic kidney disease)208. To what extent this effect might be linked to increased intake of nitrate is unknown. To date, no placebo-controlled clinical trial has investigated the effects of chronic nitrate supplementation in patients with kidney disease. However, a crossover study in patients with CKD (chronic kidney disease)(stages 2-4) due to hypertensive or diabetic nephropathies showed a significant reduction of blood pressure and renal resistive index 4h after a single dose of nitrate(300mg)2. Moreover, a prospective study with a follow-up period of almost 6 years concluded that a habitually high intake of nitrate and/or nitrite from dietary sources was independently associated with a significantly reduced risk of hypertension and CKD (chronic kidney disease)210.

Taken together, clinical studies have demonstrated that nitrate supplementation is associated with the lowering of blood pressure, which seems to be more pronounced in patients with hypertension. In healthy individuals, this effect is not associated with significant changes in kidney function, whereas favourable effects on renal haemodynamics were observed in patients with CKD (chronic kidney disease). Future longer-term, placebo-controlled, randomized trials are needed to determine if supplementation with inorganic nitrate and/or nitrite to restore NO (nitric oxide) bioactivity could be a beneficial additive treatment to slow the progression of kidney disease and associated cardiovascular and metabolic disorders. Such effects have consistently been reported in experimental studies.

CKD (chronic kidney disease): How NO (nitric oxide) affects kidney disease

Conclusions and future perspectives

Several decades have passed since the discovery of NO (nitric oxide) as the elusive endothelium-derived relaxing factor, but some controversies still exist regarding its formation and the true identity of the signalling molecule, as well as its downstream signalling and effector sites in health and disease. NO (nitric oxide) and other bioactive nitrogen oxide species have pivotal roles in multiple physiological functions, including modulation of the kidney, cardiovascular and metabolic systems. NO (nitric oxide) is classically derived from L-arginine-dependent NOS (nitric oxide synthase) isoforms, but can also be formed endogenously via serial reduction steps of inorganic nitrate and nitrite. This nitrate-nitrite-NO (nitric oxide) path-way, which can be boosted via the diet, is of particular importance in conditions where the activity of the NOS (nitric oxide synthase) system is reduced or non-functional (that is, hypoxia, ischaemia and low pH). Downstream signalling and functional effects are linked with both cGMP-dependent and -independent mechanisms. Reduced NO (nitric oxide) bioactivity due to compromised NO (nitric oxide) generation or increased metabolism has been associated with ageing and kidney, cardiovascular and metabolic disorders, which are often coupled with increased generation of ROS leading to oxidative stress. In the kidney, NO (nitric oxide) is crucially involved in autoregulation and modulation of tubular transport, which may be of importance in the development and progression of hypertension, CKD (chronic kidney disease), ischaemia-reperfusion injury and DKD. Although several experimental studies have demonstrated favourable effects of nitrate and nitrite supplementation on kidney disease and associated complications, these results await further clinical translation. Existing and future novel strategies that increase NObioactivity and reduce oxidative stress, via both pharmacological and nutritional approaches, may have therapeutic potential to prevent and treat kidney disease and associated cardiometabolic complications. One of the challenges with such novel drug candidates is to optimize their spatial and temporal delivery to achieve the desired effects without any unwanted disturbances in normal physiological redox signalling.