Endothelin blockade prevents the long-term cardiovascular and renal sequelae of acute kidney injury in mice.

Acute kidney injury (AKI) is common and associated with increased risks of cardiovascular and chronic kidney disease. Causative molecular/physiological pathways are poorly defined. There are no therapies to improve long-term outcomes. An activated endothelin system promotes cardiovascular and kidney disease progression. We hypothesized a causal role for this in the transition of AKI to chronic disease. Plasma endothelin-1 was threefold higher; urine endothelin-1 was twofold higher; and kidney preproendothelin-1, endothelin-A, and endothelin-B receptor message up-regulated in patients with AKI. To show causality, AKI was induced in mice by prolonged ischemia with a 4-week follow-up. Ischemic injury resulted in hypertension, endothelium-dependent and endothelium-independent macrovascular and microvascular dysfunction, and an increase in circulating inflammatory Ly6Chigh monocytes. In the kidney, we observed fibrosis, microvascular rarefaction, and inflammation. Administration of endothelin-A antagonist, but not dual endothelin-A/B antagonist, normalized blood pressure, improved macrovascular and microvascular function, and prevented the transition of AKI to CKD. Endothelin-A blockade reduced circulating and renal proinflammatory Ly6Chigh monocytes and B cells, and promoted recruitment of anti-inflammatory Ly6Clow monocytes to the kidney. Blood pressure reduction alone provided no benefits; blood pressure reduction alongside blockade of the endothelin system was as effective as endothelin-A antagonism in mitigating the long-term sequelae of AKI in mice. Our studies suggest up-regulation of the endothelin system in patients with AKI and show in mice that existing drugs that block the endothelin system, particularly those coupling vascular support and anti-inflammatory action, can prevent the transition of AKI to chronic kidney and cardiovascular disease.

A novel role for myeloid endothelin-B receptors in hypertension.

AIMS: Hypertension is common. Recent data suggest that macrophages (Mφ) contribute to, and protect from, hypertension. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor with additional pro-inflammatory properties. We investigated the role of the ET system in experimental and clinical hypertension by modifying Mφ number and phenotype. METHODS AND RESULTS: In vitro, Mφ ET receptor function was explored using pharmacological, gene silencing, and knockout approaches. Using the CD11b-DTR mouse and novel mice with myeloid cell-specific endothelin-B (ETB) receptor deficiency (LysMETB-/-), we explored the effects of modifying Mφ number and phenotype on the hypertensive effects of ET-1, angiotensin II (ANG II), a model that is ET-1 dependent, and salt. In patients with small vessel vasculitis, the impacts of Mφ depleting and non-depleting therapies on blood pressure (BP) and endothelial function were examined. Mouse and human Mφ expressed both endothelin-A and ETB receptors and displayed chemokinesis to ET-1. However, stimulation of Mφ with exogenous ET-1 did not polarize Mφ phenotype. Interestingly, both mouse and human Mφ cleared ET-1 through ETB receptor mediated, and dynamin-dependent, endocytosis. Mφ depletion resulted in an augmented chronic hypertensive response to both ET-1 and salt. LysMETB-/- mice displayed an exaggerated hypertensive response to both ET-1 and ANG II. Finally, in patients who received Mφ depleting immunotherapy BP was higher and endothelial function worse than in those receiving non-depleting therapies. CONCLUSION: Mφ and ET-1 may play an important role in BP control and potentially have a critical role as a therapeutic target in hypertension.

Renal and Blood Pressure Response to a High-Salt Diet in Mice With Reduced Global Expression of the Glucocorticoid Receptor.

Salt-sensitive hypertension is common in glucocorticoid excess. Glucocorticoid resistance also presents with hypercortisolemia and hypertension but the relationship between salt intake and blood pressure (BP) is not well defined. GRßgeo/+ mice have global glucocorticoid receptor (GR) haploinsufficiency and increased BP. Here we examined the effect of high salt diet on BP, salt excretion and renal blood flow in GRßgeo/+mice. Basal BP was ∼10 mmHg higher in male GRßgeo/+ mice than in GR+/+ littermates. This modest increase was amplified by ∼10 mmHg following a high-salt diet in GRßgeo/+ mice. High salt reduced urinary aldosterone excretion but increased renal mineralocorticoid receptor expression in both genotypes. Corticosterone, and to a lesser extent deoxycorticosterone, excretion was increased in GRßgeo/+ mice following a high-salt challenge, consistent with enhanced 24 h production. GR+/+ mice increased fractional sodium excretion and reduced renal vascular resistance during the high salt challenge, retaining neutral sodium balance. In contrast, sodium excretion and renal vascular resistance did not adapt to high salt in GRßgeo/+ mice, resulting in transient sodium retention and sustained hypertension. With high-salt diet, Slc12a3 and Scnn1a mRNAs were higher in GRßgeo/+ than controls, and this was reflected in an exaggerated natriuretic response to thiazide and benzamil, inhibitors of NCC and ENaC, respectively. Reduction in GR expression causes salt-sensitivity and an adaptive failure of the renal vasculature and tubule, most likely reflecting sustained mineralocorticoid receptor activation. This provides a mechanistic basis to understand the hypertension associated with loss-of-function polymorphisms in GR in the context of habitually high salt intake.

Cardiac Metabolic Deregulation Induced by the Tyrosine Kinase Receptor Inhibitor Sunitinib is rescued by Endothelin Receptor Antagonism.

The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, we explored the cardiotoxicity of the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. We showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis and reduced left ventricular ejection fraction as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of the endothelin receptor antagonist, macitentan, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostic approach.

First-in-Man Demonstration of Direct Endothelin-Mediated Natriuresis and Diuresis.

Endothelin (ET) receptor antagonists are potentially novel therapeutic agents in chronic kidney disease and resistant hypertension, but their use is complicated by sodium and water retention. In animal studies, this side effect arises from ETB receptor blockade in the renal tubule. Previous attempts to determine whether this mechanism operates in humans have been confounded by the hemodynamic consequences of ET receptor stimulation/blockade. We aimed to determine the effects of ET signaling on salt transport in the human nephron by administering subpressor doses of the ET-1 precursor, big ET-1. We conducted a 2-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. After sodium restriction, subjects received either intravenous placebo or big ET-1, in escalating dose (≤300 pmol/min). This increased plasma concentration and urinary excretion of ET-1. Big ET-1 reduced heart rate (≈8 beats/min) but did not otherwise affect systemic hemodynamics or glomerular filtration rate. Big ET-1 increased the fractional excretion of sodium (from 0.5 to 1.0%). It also increased free water clearance and tended to increase the abundance of the sodium-potassium-chloride cotransporter (NKCC2) in urinary extracellular vesicles. Our protocol induced modest increases in circulating and urinary ET-1. Sodium and water excretion increased in the absence of significant hemodynamic perturbation, supporting a direct action of ET-1 on the renal tubule. Our data also suggest that sodium reabsorption is stimulated by ET-1 in the thick ascending limb and suppressed in the distal renal tubule. Fluid retention associated with ET receptor antagonist therapy may be circumvented by coprescribing potassium-sparing diuretics.

Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

The role of smooth muscle endothelinB (ETB) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ETB receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ETB receptors were selectively deleted from smooth muscle by crossing floxed ETB mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ETB deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ETB was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ETB-mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ETB-mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ETB knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ETB blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ETB-mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ETB knockout mice. In the absence of other pathology, ETB receptors in vascular smooth muscle make a small but significant contribution to ETB-dependent regulation of BP. These ETB receptors have no effect on vascular contraction or neointimal remodeling.

Chorioretinal thinning in chronic kidney disease links to inflammation and endothelial dysfunction.

BACKGROUND. Chronic kidney disease (CKD) is strongly associated with cardiovascular disease and there is an established association between vasculopathy affecting the kidney and eye. Optical coherence tomography (OCT) is a novel, rapid method for high-definition imaging of the retina and choroid. Its use in patients at high cardiovascular disease risk remains unexplored. METHODS. We used the new SPECTRALIS OCT machine to examine retinal and retinal nerve fiber layer (RNFL) thickness, macular volume, and choroidal thickness in a prospective cross-sectional study in 150 subjects: 50 patients with hypertension (defined as a documented clinic BP greater than or equal to 140/90 mmHg (prior to starting any treatment) with no underlying cause identified); 50 with CKD (estimated glomerular filtration rate (eGFR) 8-125 ml/min/1.73 m2); and 50 matched healthy controls. We excluded those with diabetes. The same, masked ophthalmologist carried out each study. Plasma IL-6, TNF-α , asymmetric dimethylarginine (ADMA), and endothelin-1 (ET-1), as measures of inflammation and endothelial function, were also assessed. RESULTS. Retinal thickness, macular volume, and choroidal thickness were all reduced in CKD compared with hypertensive and healthy subjects (for retinal thickness and macular volume P < 0.0001 for CKD vs. healthy and for CKD vs. hypertensive subjects; for choroidal thickness P < 0.001 for CKD vs. healthy and for CKD vs. hypertensive subjects). RNFL thickness did not differ between groups. Interestingly, a thinner choroid was associated with a lower eGFR (r = 0.35, P <0.0001) and, in CKD, with proteinuria (r = -0.58, P < 0.001) as well as increased circulating C-reactive protein (r = -0.57, P = 0.0002), IL-6 (r = -0.40, P < 0.01), ADMA (r = -0.37, P = 0.02), and ET-1 (r = -0.44, P < 0.01). Finally, choroidal thinning was associated with renal histological inflammation and arterial stiffness. In a model of hypertension, choroidal thinning was seen only in the presence of renal injury. CONCLUSIONS. Chorioretinal thinning in CKD is associated with lower eGFR and greater proteinuria, but not BP. Larger studies, in more targeted groups of patients, are now needed to clarify whether these eye changes reflect the natural history of CKD. Similarly, the associations with arterial stiffness, inflammation, and endothelial dysfunction warrant further examination. TRIAL REGISTRATION. Registration number at www.clinicalTrials.gov: NCT02132741. SOURCE OF FUNDING. TR was supported by a bursary from the Erasmus Medical Centre, Rotterdam. JJMHvB was supported by a bursary from the Utrecht University. JRC is supported by a Rowling Scholarship. SB was supported by a Wellcome Trust funded clinical research fellowship from the Scottish Translational Medicine and Therapeutics Initiative, and by a Rowling Scholarship, at the time of this work. ND is supported by a British Heart Foundation Intermediate Clinical Research Fellowship (FS/13/30/29994).

Therapeutic potential of endothelin receptor antagonism in kidney disease.

Our growing understanding of the role of the endothelin (ET) system in renal physiology and pathophysiology is from emerging studies of renal disease in animal models and humans. ET receptor antagonists reduce blood pressure and proteinuria in chronic kidney disease and cause regression of renal injury in animals. However, the therapeutic potential of ET receptor antagonism has not been fully explored and clinical studies have been largely limited to patients with diabetic nephropathy. There remains a need for more work in nondiabetic chronic kidney disease, end-stage renal disease (patients requiring maintenance dialysis and those with a functioning kidney transplant), ischemia reperfusion injury, and sickle cell disease. The current review summarizes the most recent advances in both preclinical and clinical studies of ET receptor antagonists in the field of kidney disease.

25 years of endothelin research: the next generation.

In the past three decades, endothelin and endothelin receptor antagonists have received great scientific and clinical interest, leading to the publication of more than 27,000 scientific articles since its discovery. The Thirteenth International Conference on Endothelin (ET-13) was held on September 8-11, 2013, at Tokyo Campus of the University of Tsukuba in Japan. Close to 300 scientists from 25 countries from around the world came to Tokyo to celebrate the anniversary of the discovery of the endothelin peptide discovered 25 years ago at the University of Tsukuba. This article summarizes some of the highlights of the conference, the anniversary celebration ceremony, and particularly the participation of next generation of endothelin researchers in endothelin science and the anniversary celebration. As a particular highlight, next generation endothelin researchers wrote a haiku (a traditional form of Japanese poetry originating from consisting of no more than three short verses and 27 on, or Japanese phonetic units) to describe the magic of endothelin science which they presented to the conference audience at the anniversary ceremony. The text of each haiku - both in its original language together with the English translation - is part of this article providing in an exemplary fashion how poetry can be bridged with science. Finally, we give an outlook towards the next 25 years of endothelin research.

Diurnal variation in blood pressure and arterial stiffness in chronic kidney disease: the role of endothelin-1.

Hypertension and arterial stiffness are important independent cardiovascular risk factors in chronic kidney disease (CKD) to which endothelin-1 (ET-1) contributes. Loss of nocturnal blood pressure (BP) dipping is associated with CKD progression, but there are no data on 24-hour arterial stiffness variation. We examined the 24-hour variation of BP, arterial stiffness, and the ET system in healthy volunteers and patients with CKD and the effects on these of ET receptor type A receptor antagonism (sitaxentan). There were nocturnal dips in systolic BP and diastolic BP and pulse wave velocity, our measure of arterial stiffness, in 15 controls (systolic BP, −3.2±4.8%, P<0.05; diastolic BP, −6.4±6.2%, P=0.001; pulse wave velocity, −5.8±5.2%, P<0.01) but not in 15 patients with CKD. In CKD, plasma ET-1 increased by 1.2±1.4 pg/mL from midday to midnight compared with healthy volunteers (P<0.05). Urinary ET-1 did not change. In a randomized, double-blind, 3-way crossover study in 27 patients with CKD, 6-week treatment with placebo and nifedipine did not affect nocturnal dips in systolic BP or diastolic BP between baseline and week 6, whereas dipping was increased after 6-week sitaxentan treatment (baseline versus week 6, systolic BP: −7.0±6.2 versus −11.0±7.8 mm Hg, P<0.05; diastolic BP: −6.0±3.6 versus −8.3±5.1 mm Hg, P<0.05). There was no nocturnal dip in pulse pressure at baseline in the 3 phases of the study, whereas sitaxentan was linked to the development of a nocturnal dip in pulse pressure. In CKD, activation of the ET system seems to contribute not only to raised BP but also the loss of BP dipping. The clinical significance of these findings should be explored in future clinical trials.

Endothelin antagonism and its role in the treatment of hypertension.

Hypertension contributes greatly to global disease burden and in many patients current treatments do not adequately control blood pressure (BP). Endothelin-1 (ET-1) is a potent vasoconstrictor that is implicated in the pathogenesis of hypertension, including the hypertension that is often associated with chronic kidney disease (CKD) and the metabolic syndrome. ET receptor antagonists, currently licensed for the treatment of pulmonary arterial hypertension and scleroderma-related digital ulcers, are being investigated for the treatment of hypertension. Clinical trials have addressed the use of ET receptor antagonists as monotherapy in primary hypertension, as an add-on therapy in resistant hypertension and in CKD. This review will evaluate the current evidence regarding the therapeutic potential of ET receptor antagonists in hypertension, as well as highlighting important issues that still need to be addressed.

Gamma-agostic species as key intermediates in the vinyl addition polymerization of norbornene with cationic (allyl)Pd catalysts: synthesis and mechanistic insights.

Several cationic (allyl)Pd(II) complexes were synthesized and shown to be highly active for (2,3)-vinyl addition polymerization of norbornene (NB) to yield polymers with low molecular weight distributions (MWDs) ranging from 1.2-1.4. Despite the low MWDs, slow initiation was followed by rapid propagation preventing molecular weight control of the poly(norbornene). Several intermediates in these polymerizations initiated with [(2-R-allyl)Pd(mesitylene)](+) complexes were fully characterized (NMR and X-ray diffraction). Consistent with previous observations the allyl and NB units couple in cis-exo fashion to yield a sigma,pi-complex capped by mesitylene. Mesitylene is readily displaced by NB to form an agostic intermediate in which NB acts as a bidentate ligand and binds to the cationic Pd center via the pi-system and a gamma-agostic interaction with the syn hydrogen at C7. The identity of this complex was established by NMR spectroscopy and single-crystal X-ray diffraction. It is significant since it suggests bidentate binding of NB in the propagating species, which cannot be observed by NMR spectroscopy. The NMR studies suggest that the second insertion, i.e., insertion of NB in the agostic intermediate, is the slow initiation step and the subsequent insertions are extremely fast. Therefore, slow chelate opening is the major limitation preventing a living polymerization. This hypothesis was explored using a series of cationic substituted pi-allyl complexes; significantly increased reactivity was observed when electron-withdrawing groups were introduced into the allyl moiety. However, despite these modifications initiation remained slow relative to chain propagation.