J. J. Rob Hermans

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

Neuroprotection after stroke by targeting NOX4 as a source of oxidative stress.

SIGNIFICANCE Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. RECENT ADVANCES Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. CRITICAL ISSUES We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. FUTURE DIRECTIONS The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy.

Transient AT1 receptor-inhibition in prehypertensive spontaneously hypertensive rats results in maintained cardiac protection until advanced age.

Objective In young spontaneously hypertensive rats (SHR), transient angiotensin II type 1 receptor (AT1R) blockade decreases blood pressure for a prolonged period. We tested the hypothesis that transient AT1R blockade in SHR leads to cardiac protection until advanced age. Method Wistar–Kyoto rats, SHR and transiently losartan-treated SHR (SHR-Los) (20 mg/kg per day; weeks 4–8 of age) were followed up until week 72 (n = 9 each group), including repeated echocardiography, radiotelemetric investigations and 24-h urine collection. End-point measurements comprised left ventricular function parameters, left ventricular histomorphology and molecular biology (types I and III collagen, brain natriuretic peptide, AT1R mRNA) as well as renal morphology. Results Prehypertensive treatment with losartan, but not with the general vasodilator hydralazine, reduced blood pressure until age 48 weeks. In untreated SHR, the end-diastolic volume increased from week 36 and the left ventricular ejection fraction fell from week 48. In contrast, age-related changes in end-diastolic volume and ejection fraction were comparable in SHR-Los and Wistar–Kyoto rats up to age 60 weeks. At age 72 weeks, the ejection fraction was reduced in SHR-Los but higher than that in untreated SHR (ejection fraction: Wistar–Kyoto rats, 58 ± 3%; SHR, 39 ± 3%; SHR-Los, 46 ± 3%; P < 0.01 and P < 0.05, respectively). The heart weight/body weight ratio (SHR-Los, 4.7 ± 0.1 g/kg; SHR, 5.2 ± 0.2 g/kg) and cardiac brain natriuretic peptide mRNA levels were improved by treatment. Left ventricular histomorphology and 24-h albuminuria were significantly improved in SHR-Los (41 ± 5 mg/day; SHR, 80 ± 22 mg/day; P < 0.05). Conclusion In young SHR, transient AT1R blockade, not blood pressure lowering, attenuates the development of hypertension and exerts cardioprotective effects up to age 72 weeks.

Synthesis, biological evaluation, and molecular modeling of 1-benzyl-1H-imidazoles as selective inhibitors of aldosterone synthase (CYP11B2).

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, this is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, we used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-yl)methyl)benzonitrile) displaying an IC(50) for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC(50) for CYP11B2 6.0 nM, selectivity 19.8). Molecular docking of the inhibitors in the models enabled us to generate posthoc hypotheses on their binding modes, providing a valuable basis for future studies and further design of CYP11B2 inhibitors.

Transient prehypertensive treatment in spontaneously hypertensive rats: a comparison of spironolactone and losartan regarding long-term blood pressure and target organ damage.

Objective We previously demonstrated that when the renin–angiotensin system (RAS) is transiently blocked by an angiotensin receptor blocker (ARB) in young spontaneously hypertensive rats (SHR), this results in a prolonged blood pressure decrease and protection against target organ damage. Aldosterone is an essential hormone in the RAS, and contributes to pathologic remodeling. Thus, part of the protective effects of the ARB may be due to inhibition of aldosterone-mediated effects. To test this hypothesis, in young SHR, we compared the effectiveness of transient treatment with the mineralocorticoid receptor blocker spironolactone with those obtained by the ARB losartan. Methods SHR were transiently (i.e. between 4–8 weeks of age) treated with spironolactone (SHR-Spiro: 1 mg/kg per day), losartan (SHR-Los: 20 mg/kg per day) or saline. Rats were followed up until week 72 of age and cardiovascular parameters were repeatedly assessed by echocardiography, radiotelemetry of blood pressure and 24-h urine collection. End-point measurements included direct left ventricular contractility and relaxation, as well as cardiac and renal histomorphology. Results Transient spironolactone treatment reduced blood pressure up to 36 weeks of age and cardiac and renal collagen deposition and tubular atrophy up to 72 weeks of age compared to untreated SHR. Pulse pressure was higher in SHR-Spiro compared to SHR-Los. Cardiac hypertrophy, albuminuria and glomerulosclerosis were not attenuated in SHR-Spiro compared to untreated SHR up to 72 weeks of age, whereas the effects in SHR-Los were ameliorated. Conclusions Although transient spironolactone treatment leads to prolonged blood pressure reduction and reduced collagen deposition, long-term organ protection only partially exists. Thus, transient spironolactone treatment is less effective than transient losartan treatment.

Direct renin inhibition in a rat model of chronic allograft injury.

Background. Blockade of the renin-angiotensin system (RAS) with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor blockers (ARBs) slow the progression of various chronic kidney diseases and chronic allograft dysfunction. RAS inhibition can be achieved also by directly blocking renin upstream from ACE. However, direct renin inhibition can have additional effects since formation of renoprotective Ang II breakdown products such as angiotensin (Ang) (1–7) that are produced by ACE2 are also inhibited. Methods. Using a Fischer-to-Lewis renal transplantation model, the effect of the renin inhibitor aliskiren (10 mg/kg/day) was assessed on the development of chronic allograft dysfunction compared with vehicle treatment and Ang II receptor blockers candesartan. Results. Aliskiren had no effect on renal function (proteinuria, creatinine clearance) or on renal morphological changes (glomerulosclerosis collagen deposition, myofibroblast accumulation and macrophage infiltration) compared with the vehicle- and candesartan-treated animals determined 24 weeks after transplantation. On the other hand, atrophy of tubular cells was significantly attenuated. Candesartan reduced both proteinuria and structural injury of the kidney. In aliskiren-treated animals reduced serum Ang II and Ang (1–7) levels were detected, whereas the level of urine angiotensinogen was unchanged. Conclusions. The renin inhibitor aliskiren does not slow the progression of chronic allograft dysfunction. We suggest that the lack of protection might be due to reduced formation of the protective Ang II breakdown products such as Ang (1–7) or due to unchanged intrarenal RAS activity demonstrated by urinary angiotensinogen levels.

Functional and Structural Postglomerular Alterations in the Kidney of Prehypertensive Spontaneously Hypertensive Rats

The kidney plays a major role in the development of hypertension. Following the Borst‐Guyton theory of an altered set‐point for fluid and electrolyte homeostasis we aim to investigate functional and structural renal parameter during the development of hypertension. Therefore we focus on counter current exchange related factors. We compared 4 and 8 weeks old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) concerning basic renal parameters as creatinine and phosphorus clearance and urinary osmolality. Mean arterial pressure (MAP) was measured intra‐arterially. Vasa recta were investigated using immunohistochemistry for α‐smooth‐muscle actin (ASMA) and plastification for geometric analyses. Blood pressure was not yet significantly elevated in SHR at 4 weeks but at 8 weeks it was higher in SHR (116 ± 7 vs. 102 ± 4 mm Hg; p < 0.01). Kidney weight/body weight ratio was lower in SHR at both ages. In 4 weeks old SHR, phosphorus clearance and urinary osmolality were decreased compared to WKY [0.02 ± 0.01 vs. 0.05 ± 0.02 (ml/min*100 g BW) p < 0.03; 14.2 ± 2.2 vs. 18.9 ± 2.9 (osmol/kg*24 h urine) p < 0.05] indicating reduced tubular reabsorption. At 8 weeks phosphorus clearance and urinary osmolality were comparable to WKY. α‐Actin was found in vasa recta in a 4‐times higher degree in SHR with a predominant location in the outer medulla. Radii of vasa recta in the outer medulla decreased during development. In plastificated sections vasa recta of SHR revealed sphincter‐like pattern. Functional and structural alterations related to the counter current exchanger are already evident in prehypertensive SHR. During development of hypertension both factors get adapted to higher blood pressure level. Sphincter‐like structures in vasa recta suggest contractility of pericytes/vascular smooth muscle cells (vSMC). As these were just seen in SHR that might allude to a higher potential to contract. We conclude that differences in postglomerular structure and function may contribute to the development of hypertension in SHR.

Effect of angiotensin II on rat renal cortical 11β-hydroxysteroid dehydrogenase

Renal 11β-hydroxysteroid dehydrogenases (11β-HSDs) are subject to modulation by various endogenous factors. 11β-HSDs convert glucocorticoids into inactive 11-ketones and thereby determine tissue levels of active glucocorticoids and thus the extent of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation. As such, modulation of the activity of renal 11β-HSDs may contribute to the cascade of regulatory events involved in renal electrolyte water handling. We investigated whether renal 11β-HSDs are modulated by elevated circulating angiotensin II. In rats infused for 2 wk with angiotensin II (250 ng/[kg.min] subcutaneously), plasma angiotensin II, aldosterone, and corticosterone were raised 5.1-, 10.7-, and 2.3-fold, respectively, compared with control rats. Angiotensin II infusion raised corticosterone 11β-oxidation 1.46- and 1.35-fold in renal cortical proximal and distal tubules (enriched by Percoll centrifugation), respectively, but had no effect on 11β-HSD1 and 11β-HSD2 mRNA levels (semiquantitative reverse transcriptase solymerase chain reaction), except for distal tubular 11β-HSD1 mRNA, which was decreased to 50%. In vitro treatment of freshly isolated tubules with angiotensin II for 45 min prior to assessment of 11β-HSD activity showed no direct acute effects of angiotensin II on tubular corticosterone 11β-oxidation. The enhanced renal tubular corticosterone 11β-oxidation in vivo may partly protect renal GR and MR from elevated plasma corticosterone on angiotensin II infusion.

Comparison of the effects of intrapericardial and intravenous aldosterone infusions on left ventricular fibrosis in rats.

Aldosterone plays a detrimental role in the pathology of chronic heart failure. An important mechanism resides in its ability to evoke extensive fibrosis in the heart. However, the locations of the aldosterone interaction sites responsible for triggering cardiac fibrosis are puzzling. Extra‐cardiac aldosterone actions are known to contribute to cardiac fibrosis but whether local cardiac aldosterone actions are involved is unclear.

Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform?

We studied 11β-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1 and the effects of different steroids on them. Cortisol was oxidized in the presence of NAD as well as NADP, reflecting the presence of two different 11β-HSD forms. Enzyme kinetics for cortisol 11β-oxidation were: Vmax=5.9 pmol/(min×mg), Km=0.2 μM with NAD, and Vmax=4.5 pmol/(min×mg), Km=1.0 μM with NADP. Interestingly, no reverse reaction was observed when using cortisone and NADPH as substrate and cosubstrate, respectively. Exposure of cells to a variety of steroids had different effects on cortisol 11β-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3–60 min of exposure) decreased the NAD-dependent 11β-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11β-oxidation of cortisol with NADP increased by dexamethasone treatment of the cells, after a lagtime of about 2 h, and this effect was still evident after 32 h. The increase of 11β-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC50: 125 nM). The antiglucocorticoid RU 486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 μM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11β-oxidation, but had no effect on the NAD-dependent 11β-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11β-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK1 cells, two forms of 11β-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11β-HSD 2, the NADP-dependent 11β-HSD possibly resembles an as yet unknown third isoform.