Nitric oxide signalling in kidney regulation and cardiometabolic health

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 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 synthase (NOS) pathway is classically viewed as the main source of endogenous NO formation. However, the function of the NOS system is often compromised in various pathologies including kidney, cardiovascular and metabolic diseases. An alternative pathway, the nitrate-nitrite-NO pathway, enables endogenous or dietary-derived inorganic nitrate and nitrite to be recycled via serial reduction to form bioactive nitrogen species, including NO, independent of the NOS system. Signalling via these nitrogen species is linked with cGMP-dependent and independent mechanisms. Novel approaches to restoring NO homeostasis during NOS deficiency and oxidative stress have potential therapeutic applications in kidney, cardiovascular and metabolic disorders.

Fig. 1. The NOS pathway and potential effects of NO on cardiovascular, renal and metabolic functions.

Nitric oxide (NO) is endogenously formed by three different nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The activity of these enzymes is oxygen dependent and requires l-arginine and several co-factors (calmodulin, nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin (BH₄), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)). NO binds to the reduced heme site (Fe²⁺) of soluble guanylyl cyclase (sGC), which activates this enzyme, leading to the formation of the second messenger cyclic GMP (cGMP) from GTP. NO is a short-lived molecule that is oxidized in blood and tissues to form nitrite (NO₃⁻), nitrate (NO₂⁻) and other bioactive nitrogen species. NO bioactivity has been associated with numerous favourable effects in cardiovascular, renal and metabolic systems, mainly via cGMP-dependent mechanisms, although cGMP-independent mechanisms have also been reported. These mechanisms are multifactorial and involve modulation of protein function and immune cells, reductions in angiotensin II (Ang II) signalling, oxidative stress and sympathetic nerve activity and modulation of mitochondrial function. GFR, glomerular filtration rate.

Fig. 2. The generation of bioactive NO in mammals.

Nitric oxide (NO) is classically viewed to be formed via the NO synthase (NOS) pathway but can also be generated via a fundamentally different mechanism, the nitrate (NO₃⁻)–nitrite (NO₂⁻)–NO pathway. During conditions of normal oxygen tension and pH, NO and other bioactive nitrogen species are oxidized to form inorganic nitrite and nitrate in the blood and tissues. Circulating NO₃⁻ and NO₂⁻ can be reduced back to NO and other bioactive nitrogen species via non-enzymatic and enzymatic systems. This alternative pathway of NO generation is of particular importance during low oxygen tension (that is, ischaemia and hypoxia) and acidic conditions. In addition to NOS-derived NO₃⁻, which is formed following oxidation of NO, dietary inorganic nitrate is a major contributor to the pool of this anion in the body. In particular, green leafy vegetables and beetroot contain high levels of inorganic nitrate. Commensal oral bacteria are crucial for the reduction of NO₃⁻ to NO₂⁻, whereas conversion of NO₂⁻ to NO occurs in the acidic milieu of the stomach and in the circulation as a result of non-enzymatic and enzymatic systems (for example, deoxyhaemoglobin (deoxy-Hb), deoxymyoglobin (deoxy-Mb), xanthine oxidoreductase (XOR) and mitochondrial complexes). eNOS, epithelial NOS; iNOS, inducible NOD; nNOS, neuronal NOS.

Fig. 3. cGMP-independent signalling via bioactive nitrogen species.

The nitric oxide synthase (NOS) systems and serial reductions of nitrate (NO₃⁻) and nitrite (NO₂⁻) lead to the formation of nitric oxide (NO^•) and other bioactive nitrogen species. These species can undergo nitration or nitrosation/nitrosylation reactions independent of cyclic GMP (cGMP) signalling and modify proteins, lipids, nucleosides and metals as well as induce transnitration, which can alter gene expression, receptor signalling, enzyme activity and mitochondrial function and elicit antioxidant, anti-inflammatory, antifibrotic and inotropic effects. DNIC, dinitrosyl−iron complexe; eNOS, epithelial NOS; heme-NO, nitrosyl-heme; iNOS, inducible NOS; N₂O₃, dinitrogen trioxide; nNOS, neuronal NOS; NO₂^•, nitrogen dioxide; ONOO⁻, peroxynitrite; SNO, S-nitrosothiols.

Fig. 4. Effects of NO on sodium transporters in the nephron.

Nitric oxide (NO) is generally considered to inhibit tubular sodium reabsorption along the nephron. However, differing results have been obtained in acute and chronic conditions, in different experimental settings (in vivo versus ex vivo or in vitro) and in different species. Moreover, the effects of NO on tubular sodium (Na⁺) handling seem to be dependent on hormonal activity, particularly via interaction with the renin–angiotensin–aldosterone system. In the proximal tubule, neuronal NO synthase (nNOS) and endothelial NOS (eNOS)-derived NO has been reported to inhibit the basolateral sodium–potassium pump (Na⁺/K⁺-ATPase) and the apical sodium/hydrogen exchanger 3 (NHE3), as well as to modulate the activity of the basolateral Na⁺/HCO₃⁻ cotransporter. In the thick ascending limb (TAL) of the loop of Henle, eNOS-derived NO inhibits NHE3 and may also inhibit the apical Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2). eNOS-derived NO also inhibits NKCC2 in macula densa cells. Activation of nNOS in the macula densa can inhibit paracrine signalling mediated via adenosine triphosphate (ATP) and adenosine (ADO), which forms part of the tubuloglomerular feedback mechanism following activation of purinergic P₂ and/or adenosine A₁ receptors located on vascular smooth muscle cells in the afferent arteriole. nNOS expression has been demonstrated in the distal tubule but the potential effects of NO on specific transporters in this segment of the nephron (for example, the Na⁺/Cl⁻ cotransporter) are currently not clear. Finally, in collecting duct cells, nNOS-derived NO can inhibit the epithelial sodium channel (ENaC).

Fig. 5. Strategies to restore NO bioactivity.

Endogenous nitric oxide (NO) bioactivity originates from the classical NO synthase (NOS) pathway and the alternative nitrate (NO₃⁻)–nitrite (NO₂⁻)–NO pathway. Signalling via NO and other bioactive nitrogen species involves soluble guanylate cyclase (sGC)-dependent formation of cyclic GMP (cGMP) as well as cGMP-independent effects. Kidney, cardiovascular and metabolic disorders are associated with reduced NO production and/or signalling, which might result from increased production of reactive oxygen species (ROS) from NADPH oxidase (NOX), mitochondria or uncoupled eNOS owing to substrate or co-factor deficiency. Several approaches might increase NO bioactivity. First, increasing or restoring endogenous NOS activity by supplementing with l-arginine, l-citrulline or tetrahydrobiopterin (BH₄), inhibiting arginase or blocking the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA). Statins, novel modulators of the renin–angiotensin–aldosterone system (RAAS) and compounds that increase hydrogen sulfide (H₂S) formation or signalling might also restore NOS activity. Second, inhalation of NO or inorganic NO₂⁻, treatment with organic nitrates that directly or indirectly increase NO generation independent of the NOS system or dietary supplementation with inorganic nitrate. Compared with inhalation of NO and organic nitrates, the dietary approach, using inorganic nitrate, has a more favourable pharmacokinetic and pharmacodynamic profile and is associated with lower risk of tolerance and adverse effects. Third, limiting NO metabolism, for example, by reducing the generation of ROS and thereby preventing scavenging of NO, for example, using novel antioxidants and NOX inhibitors. In addition, angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 receptor (AT₁) antagonists, which block angiotensin II (Ang II) formation and signalling, respectively, might reduce mitochondrial and NOX-derived ROS generation. Another novel approach is to oppose classical Ang II signalling by targeting the ACE2−neutral endopeptidase (NEP)−Ang(1−7)−AT₂−Mas receptor pathway using an ACE2 stimulator, AT₂ receptor agonist or Mas receptor agonist. Activation of this pathway has been associated with increased eNOS function. Finally, NO signalling can be facilitated by modulating downstream signalling pathways using phosphodiesterase 5 (PDE5) inhibitors, which inhibit the breakdown of cGMP, sGC modulators (both stimulators and activators) or compounds that increase H₂S signalling. R-ONO₂, nitrate ester.