Hipótesis especificas

All three isoforms of nitric oxide synthase (NOS) produce NO by a 5 electron oxidation of a guanidine group of its substrate, L-arginine. NOS activity is reliant on molecular oxygen and NADPH as co-substrates, and tetrahydrobiopterin (BH4), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) as cofactors of the reaction. NO production requires electron transfer from NADPH bound at the C-terminal, to a haem moiety at the N-terminal, via the flavin groups. Another electron is also probably donated by BH4. At the haem group, molecular oxygen is reduced and activated, and used for the oxidation of L-arginine, firstly to hydroxyl-L-arginine, and then to L-citrulline and NO (Förstermann and Sessa, 2012). Endothelial nitric oxide synthase (eNOS) is the principle isoform of NOS within the cardiovascular system, present in ECs, cardiac myocytes, renal tubular epithelial cells and platelets. Under physiological conditions, eNOS constitutively produces vascular NO, which is involved in vasodilatation, angiogenesis, platelet and inflammatory cell activation, and other aspects of vascular function.

In certain circumstances, the reduction of molecular oxygen and haem becomes ‘uncoupled’ with the production of NO, such that O2- is produced instead. When this occurs, uncoupled eNOS contributes to oxidative stress by reduced production of NO, and increased production of O2- and its downstream products such as H2O2 and peroxynitrite (Forstermann et al., 1994). There is evidence that eNOS uncoupling is involved in oxidative stress and endothelial dysfunction in the presence of a range of typical cardiovascular risk factors including dyslipidaemia (Stroes et al., 1997), hypertension (Higashi et al., 2002) and diabetes (Heitzer et al., 2000).

A number of mechanisms are thought to be involved with eNOS uncoupling. Depletion of the essential cofactor BH4 is one putative mechanism behind eNOS uncoupling, suggested by the fact that NO production by eNOS correlates with intracellular concentrations of BH4 (Werner-Felmayer et al., 1993), and that supplementation with BH4 increases NO production (Pieper, 1997). BH4 concentration is dependent on the balance between production by and degradation, as well as the degree of ‘recycling’ between BH4 and its oxidised metabolites. Factors which influence this include the presence of ROS and derivatives; for example,

peroxynitrite causes oxidation of BH4 to BH3 and BH2, which have no cofactor activity with eNOS (Kuzkaya et al., 2003). In a mouse model of accelerated atherosclerosis, treatment with peroxynitrite results in strikingly depleted BH4 concentrations, and increased ROS production in a manner dependent on eNOS expression (Laursen et al., 2001).

L-arginine deficiency is another mechanism by which eNOS uncoupling can occur. That said, the normal plasma concentration of L-arginine is around 100 µmol/L, whilst the level below which NO production is significantly reduced is around 3 µmol/L, such that one might not expect L-arginine deficiency to play a role in eNOS uncoupling in vivo (Closs et al., 2000). Nevertheless, L-arginine supplementation has been shown to increase NO production, and improve endothelial function in hypertension and dyslipidaemia (Imaizumi et al., 1992).

Asymmetric dimethyl-L-arginine (ADMA) is a circulating inhibitor of eNOS, and can also cause eNOS uncoupling at high levels. The enzymes involved in both production and degradation of ADMA are redox sensitive such that ADMA levels are elevated in oxidative stress. Furthermore, ADMA is excreted by the kidneys and is elevated in renal impairment. ADMA is considered separately in section 1.4.2.5.

Finally, redox modification of eNOS can also lead to uncoupling. In conditions of oxidative stress, s-glutathionylation of thiol groups occurs and can alter enzyme function. S- glutathionylation of cysteine residues within the reductase domain results in eNOS uncoupling. Arteries from spontaneously hypertensive rats (SHR) show eNOS s- glutathionylation, associated with reduced NO and increased ROS production. This is associated with reduced endothelial dependent vasodilatation, and is ameliorated by reversal of s-glutathionylation by thiol specific reducing agents (Chen et al., 2010).

Therefore, a number of mechanisms contribute to eNOS uncoupling, and given that renal impairment is associated with increased serum ADMA, oxidative stress, and antioxidant deficiency, it is likely that these mechanisms contribute to ROS production in CKD. Evidence for this comes from the fact that 5/6 nephrectomised rats showed reduced oxidative stress, and increased endothelial dependent vasodilatation when supplemented with BH4 and L-arginine; supplementation with both of these showed an additive effect (Arellano- Mendoza et al., 2011).

1.4.2.5 ADMA

ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are by-products of the methylation of arginine residues on cellular proteins, a common translational modification resulting from the action of protein arginine methyltransferases (PRMT). PRMT-1 is found in a number of cells in the cardiovascular system, and symmetrically methylates arginine to produce ADMA which is released from cells via specific transporters. Methylated arginines are eliminated by urinary excretion and, in the case of ADMA, metabolised by dimethylarginine dimethylaminohydrolase (DDAH). In renal impairment, plasma ADMA levels are elevated due to reduced urinary excretion, as well as by inhibition of DDAH by hyperhomocysteinaemia (Tarnow et al., 2004). In states of oxidative stress, PRMT-1 is upregulated, and DDAH downregulated, which is another mechanism by which ADMA is increased in CKD. ADMA inhibits all isoforms of NOS, and also causes eNOS uncoupling resulting in O2- production, and is therefore an important mediator of oxidative stress (Sydow and Munzel, 2003).

ADMA has been identified as a marker of endothelial dysfunction and cardiovascular outcomes. Cardiovascular mortality and event rate has been shown to be correlated to plasma ADMA concentration in a number of settings including atrial fibrillation (Xia et al., 2008) and after coronary angioplasty (Cavusoglu et al., 2009). Furthermore, ADMA has been found to be associated with cardiovascular event rate in chronic kidney disease, as well as in haemodialysis patients (Zoccali et al., 2001) and those with kidney transplants (Frenay et al., 2015). For example, in a study of 820 patients with stage 3 and 4 CKD, a one standard deviation increment in serum ADMA was associated with a 19% increase in cardiovascular mortality (Lu et al., 2011).