Strategies to reverse endothelial dysfunction in diabetic nephropathy

Abstract

Endothelial dysfunction underlies the basic pathophysiology of microvascular complications of diabetes. Endothelial dysfunction is associated with impaired nitric oxide (NO) availability. Since NO production is tightly regulated by endothelial nitric oxide synthase (eNOS), several therapeutic strategies have been investigated and proposed to improve eNOS bioavailability in the vasculature. The findings of Cheng et al. suggest that increased availability of eNOS may be an effective strategy in restoring endothelial function in patients with diabetic nephropathy.

The critical role of endothelial dysfunction in micro- and macrovascular complications of diabetes has generated considerable interest in identifying strategies to improve endothelial function in the diabetic milieu. An interesting study by Cheng et al.¹ confirms the importance of endothelial nitric oxide synthase (eNOS) activity in endothelial dysfunction, and reports that improving eNOS activity ameliorates progression of diabetic nephropathy. This work provides evidence for a key role of endothelial dysfunction in the progression of diabetic nephropathy, and supports a rationale for pharmacological targeting of the eNOS pathway as a novel strategy in the treatment of diabetic kidney disease.

The endothelium is not only a monolayer of endothelial cells that lines the entire vascular system, but also exerts significant autocrine, paracrine, and endocrine actions modulating vasodilation, smooth muscle cell growth and migration, and inflammatory responses. Many of these effects are mediated by nitric oxide (NO). NO opposes the effects of endothelium-derived vasoconstrictors such as angiotensin II and endothelin, protects against endothelial-cell damage induced by cytokines such as tumor necrosis factor, and provides antithrombogenic effects by blocking the release of von Willebrand factor. Indeed, a defect in the production or activity of NO promotes key features of endothelial dysfunction, such as vasoconstriction, platelet aggregation, smooth muscle cell proliferation and migration, leukocyte adhesion, and oxidative stress.

NO is formed in endothelial cells from its precursor l-arginine (l-arg) via the enzymatic action of endothelial NO synthase (eNOS), which is localized at the plasma membrane caveolae. The protein caveolin-1 binds to calmodulin to inhibit the activity of eNOS; the binding of calcium to calmodulin displaces caveolin-1, leading to activation of eNOS and increased production of NO. Cofactors such as tetrahydrobiopterin (BH₄) and nicotinamide adenine dinucleotide phosphate (NADPH) are important regulators that contribute to NO production. Optimal concentrations of BH₄ are important for the normal function of eNOS, and the depletion of BH₄ may result in ‘uncoupling’ of eNOS with a subsequent increase in superoxide production instead of NO. At the molecular level, eNOS (a 1203-amino acid, 133-kDa protein encoded by NOS3) has a bidomain structure and functions as a dimer. Within each monomer, an NH₂-terminal oxygenase domain binds a prosthetic heme group, BH₄, oxygen, and the substrate l-arg and supports the catalytic activity. A peptide sequence containing a consensus calmodulin-binding site links the oxygenase domain to a COOH-terminal reductase domain containing binding sites for flavin adenine dinucleotide, flavin mononucleotide, and NADPH. The active dimeric form of eNOS is phosphorylated at serine 1179, whereas phosphorylation at threonine 495 decreases eNOS activity. The molecular basis of eNOS signaling is shown in greater detail in Figure 1.

Figure 1. The role of eNOS in endothelial dysfunction.

NO is produced by the action of eNOS on l-arginine. This reaction requires a number of cofactors, including tetrahydrobiopterin (BH₄) and nicotinamide adenine dinucleotide phosphate (NADPH). Under physiological conditions, eNOS is phosphorylated at Ser1179 and forms homodimers. However, under conditions associated with endothelial dysfunction, eNOS can be dephosphorylated and eNOS dimerization can be compromised. Potential therapeutic options for treating endothelial dysfunction by modulating eNOS activity include administration of l-arginine or its essential cofactor BH₄ or sepiapterin (a BH₄ analog).

Endothelial dysfunction and decreased bioavailability of NO have long been proposed as some of the earliest signs of diabetic microangiopathy and major contributors to the progression of diabetic nephropathy. Indeed, one of the earliest connections of endothelial dysfunction to the pathogenesis of diabetic nephropathy was made nearly 30 years ago when Jensen et al. described increased plasma concentrations of von Willebrand factor and tissue plasminogen activator, both markers of endothelial damage, in patients with diabetic nephropathy.^2,3 Since then, various hypotheses have been put forward to explain the adverse effects of diabetes on the endothelium. Some possible targets of dysregulation leading to impaired bioavailability of NO in the diabetic milieu include advanced glycation end products, alterations in the cellular redox state by an altered NADH/NAD⁺ ratio, dysregulation of protein kinase C, and the accumulation of sorbitol. Interestingly, oxidative stress can interfere with the production and activity of NO by a number of mechanisms that are independent of hyperglycemia. For example, the free radical superoxide anion rapidly inactivates NO and destroys BH₄, a cofactor required for NO synthesis. Even when NO is produced properly, it can subsequently be rapidly inactivated by O₂⁻, particularly under conditions of high oxidative stress. In addition, hyperglycemia may influence endothelial homeostasis indirectly through increased synthesis of growth factors, in particular transforming growth factor-β and vascular endothelial growth factor. Insulin resistance is also believed to promote endothelial dysfunction. Deficiency of endothelium-derived NO is believed to be the primary defect linking insulin resistance and endothelial dysfunction.

The development of eNOS-null (eNOS^−/−) mice was a major advancement in further understanding the impact of eNOS activity on kidney function. eNOS^−/− mice exhibit an increase in blood pressure and display signs of endothelial dysfunction.⁴ This evidence served as a strong indicator of the importance of readily available NO to maintain normal endothelial function. A natural extension of these studies was to investigate the role of eNOS in the context of diabetes and its major microvascular complications. Importantly, mice with diabetes-induced eNOS deficiency exhibited significant worsening of albuminuria and histological changes consistent with human diabetic nephropathy, including those typically not seen in other mouse models of diabetic nephropathy, such as Kimmelstiel–Wilson-like nodules and arteriolar hyalinosis.^5,6 The dramatic histological changes observed in eNOS^−/− mice with type 1 and type 2 models of diabetes helped to position eNOS as a critical factor contributing to the progression of diabetic kidney disease.

Not surprisingly, pharmacological activation of the eNOS pathway has emerged as an attractive approach to ameliorate endothelial dysfunction and progression of diabetic nephropathy. In this regard, a variety of strategies have been proposed with varying degrees of success. For instance, statins have been shown to increase the stability of eNOS through their pleiotropic effects by blocking the generation of geranyl-geranyl pyrophosphate with the subsequent inhibition of the Rho–Rho kinase pathway.⁷ The role of antioxidants has also been investigated as possible therapy for endothelial dysfunction. Production of oxygen-free radicals in the vessel wall is thought to quench eNOS activity, leading to endothelial dysfunction. However, the effectiveness of antioxidants for endothelial dysfunction is not fully established,⁸ and further research is clearly needed.

Alterations in BH₄-dependent eNOS activity have also been implicated in diabetic endothelial dysfunction. eNOS has been shown to generate superoxide that is critically controlled by BH₄ levels.⁹ This NADPH-dependent formation of superoxide anion by eNOS occurring at low levels of BH₄ has been referred to as ‘uncoupling’ of NOS activity, where instead of NO production, eNOS generates superoxide anions. Notably, administration of BH₄ or precursors such as sepiapterin has been reported to improve endothelial dysfunction. One interpretation of these findings is that the increase in oxidative stress that appears to accompany most, if not all, disease processes associated with endothelial dysfunction may potentially be a result of inadequate BH₄ concentrations within the vasculature, with ensuing uncoupling of eNOS, increased superoxide formation, and diminished NO formation.

The work by Cheng et al.¹ (this issue) provides a fresh look at supplementation with l-arg and sepiapterin (a BH₄ analog) as a potential therapeutic strategy for restoring endothelial function and ameliorating progression of diabetic nephropathy. The authors initially examined the effect of l-arg and sepiapterin supplementation in the presence of high glucose on glomerular endothelial cells. They observed a significant decrease in permeability and cellular apoptosis. This was attributed to an increase in eNOS activity, as seen by a correction in eNOS dimerization, serine 1179 phosphorylation, and nitrate/nitrite synthesis (NO metabolism end products). The authors then confirmed their in vitro findings by allocating db/db mice with advanced diabetic nephropathy (age 26–34 weeks) to either l-arg or sepiapterin supplementation for 8 weeks. They found a significant reduction in glomerular basement membrane thickness and improved albuminuria. Interestingly, there was no change in mesangial matrix deposition in treated versus control animals, implying an eNOS-independent mechanism involved in mesangial matrix expansion.

The report by Cheng et al.¹ provides strong evidence on the effectiveness of therapies targeting impaired eNOS activity and thereby endothelial dysfunction in experimental models of diabetic nephropathy. This study, however, raises a number of interesting questions. For instance, would combination therapy with l-arg and sepiapterin show further improvement in endothelial dysfunction and amelioration of progression of diabetic nephropathy? Is l-arg or sepiapterin supplementation beneficial when coupled with statins or other vasoprotective modalities commonly used for endothelial dysfunction? And finally, although these findings are in agreement with a number of studies reporting on the beneficial effects of chronic BH₄ supplementation on endothelial function, long-term exposure to BH₄ has also been reported to result in a further derangement of endothelial function, possibly because BH₄ itself can enter the redox cycle and generate superoxide.¹⁰ Whether these differences are due to unrecognized secondary reactions of sepiapterin as opposed to BH₄ itself or due to a bimodal dose-dependent therapeutic efficiency of BH₄ remains unknown. Further studies are necessary to establish the real effectiveness of therapies geared specifically to target endothelial dysfunction in patients with diabetic nephropathy. These findings, if confirmed in clinical trials, will be an important advance in developing new drugs for the treatment of diabetic nephropathy and will place restoring eNOS activity and improving endothelial dysfunction at the forefront of strategies to prevent and treat microvascular complications of diabetes.