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Hypertension is a major risk factor for the pathogenesis of vascular dementia and Alzheimer’s disease. Chronic activation of the renin-angiotensin system (RAS) contributes substantially to neuroinflammation. We propose that neuroinflammation arising from chronic RAS activation can initiate and potentiate the onset of hypertension and related dementia. Neuroinflammation induced by chronic activation of the RAS plays a key role in the pathogenesis of dementia. Increased levels of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and transforming growth factor (TGF)-β have been reported in brain tissue of vascular dementia patients and animal models of vascular dementia induced by either angiotensin II infusion or transverse aortic coarctation. It is proposed that neuronal cell death and synaptic dysfunction induced by neuroinflammation lead to cognitive impairment in dementia. The neuroprotective RAS pathway, regulated by angiotensin-converting enzyme 2 (ACE2) which converts angiotensin II into angiotensin-(1–7), can attenuate hypertension and dementia. Furthermore, the use of anti-hypertensive medications in preventing dementia or cognitive decline in hypertensive patients and animal models of dementia have mostly been beneficial. Current evidence suggests a strong link between RAS induced neuroinflammation and the onset of hypertension and dementia, which warrants further investigation. Strategies to counteract an overactive RAS and enhance the neuroprotective arm of the RAS may help prevent or improve cognitive impairment associated with hypertension.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson’s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson’s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation utilising a model of human monocyte-derived microglia. We identified that SARS-CoV-2 isolates can bind and enter microglia, triggering inflammasome activation in the absence of viral replication. Mechanistically, microglial NLRP3 could be both primed and activated with SARS-CoV-2 spike glycoprotein in a NF-κB and ACE2-dependent manner. Notably, virus- and spike protein-mediated inflammasome activation in microglia was significantly enhanced in the presence of α-synuclein fibrils, which was entirely ablated by NLRP3-inhibition. These results support a possible mechanism of microglia activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in certain COVID-19 infected individuals, and a potential therapeutic avenue for intervention.SIGNIFICANCE STATEMENTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) principally affects the lungs, however there is evidence that the virus can also reach the brain and lead to chronic neurological symptoms. In this study, we examined the interaction SARS-CoV-2 with brain immune cells, by using an ex-vivo model of human monocyte-derived microglia. We identified robust activation of the innate immune sensor complex, NLRP3 inflammasome, in cells exposed to SARS-CoV-2. This was dependent on spike protein-ACE2 receptor interaction and was potentiated in the presence of α-synuclein. We therefore identify a possible mechanism for SARS-CoV-2 and increased vulnerability to developing neurological dysfunction. These findings support a potential therapeutic avenue for treatment of SARS-CoV-2 driven neurological manifestations, through use of NLRP3 inflammasome or ACE2 inhibitors.

Despite current therapies for diabetic nephropathy, many patients continue to progress to end-stage renal disease requiring renal replacement therapy. While the precise mechanisms underlying diabetic nephropathy remain to be determined, it is well established that chronic activation of the renin angiotensin aldosterone system (RAAS) plays a substantial role in the pathogenesis of diabetic nephropathy. Angiotensin converting enzyme 2 (ACE2), the enzyme responsible for activating the reno-protective arm of the RAAS converts angiotensin (Ang) II into Ang 1-7 which exerts reno-protective effects. Chronic RAAS activation leads to kidney inflammation and fibrosis, and ultimately lead to end-stage kidney disease. Currently, angiotensin converting enzyme inhibitors and Ang II receptor blockers are approved for renal fibrosis and inflammation. Targeting the reno-protective arm of the RAAS should therefore, provide further treatment options for kidney fibrosis and inflammation. In this review, we examine how targeting the reno-protective arm of the RAAS can ameliorate kidney inflammation and fibrosis and rescue kidney function in diabetic nephropathy. We argue tissue ACE2 stimulation provides a unique and promising therapeutic approach for diabetic nephropathy.

Heart failure (HF) is associated with impaired L-arginine transport. In the present study, we tested the hypothesis that augmented L-arginine transport prevents the loss of kidney function in HF. Renal function was assessed in wildtype mice (WT), transgenic mice with HF (dilated cardiomyopathy, DCM) and double transgenic mice (double transgenic mice with DCM and CAT-1 overexpression, HFCAT-1) with HF and endothelial-specific overexpression of the predominant L-arginine transporter, cationic amino acid transporter-1 (CAT-1) (n=4-8/group). Cardiac function was assessed via echocardiography and left ventricular catheterisation. Renal function was assessed via quantification of albuminuria and creatinine clearance. Plasma nitrate and nitrite levels together with renal fibrosis and inflammatory markers were also quantified at study end. Albumin/creatinine ratio was two-fold greater in DCM mice than in WT mice (P=0.002), and tubulointerstitial and glomerular fibrosis were approximately eight- and three-fold greater, respectively, in DCM mice than in WT mice (P≤0.02). Critically, urinary albumin/creatinine ratio and tubulointerstitial and glomerular fibrosis were less in HFCAT-1 mice than in DCM mice (P

Renal dysfunction and heart failure commonly co-exist; it is termed the cardiorenal syndrome (CRS). This combination of renal and cardiac impairment presents a substantial clinical challenge and is associated with adverse prognosis. The pathogenesis of the CRS is complex, including chronic activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system, together with reduced renal perfusion. Chronic activation of the RAAS can impair mitochondrial function, and increase mitochondrial derived oxidative stress which in turn can lead to renal injury and sodium and water retention. For example, it has been shown that exogenous Ang II augments renal mitochondrial oxidative stress, reduces GFR and induces albuminuria in rats with heart failure. Administration of Ang II also augmented renal mitochondrial dysfunction in aged mice. Current treatments for CRS, including angiotensin-converting enzyme inhibitors, exert limited renal protection if any at all. Therefore, novel treatments particularly those that can target renal mechanisms downstream to chronic activation of the renal renin-angiotensin system are likely to exert renoprotection in the setting of CRS.

Obesity and its complications represent one of the major emerging challenges for the developed world.1,2 Hypertension is a common sequelae of obesity,3,4 and the obesity pandemic is estimated to contribute to 75% of cases of hypertension in men and 65% in women.1 Frequently, subjects with obesity are resistant to standard antihypertensive medication,4 and poor understanding of the precise mechanisms underlying the link between obesity and hypertension has presented roadblocks for the development of new and effective therapy.4 In this context, we recently found that obesity is associated with reduced nitric oxide (NO) bioavailability caused by impaired transport of its substrate l-arginine and that augmentation of endothelial l-arginine transport prevents experimental obesity-induced hypertension.5 Findings by others also provide clear evidence that endothelial dysfunction plays an important role in the pathogenesis of hypertension6,7 including that associated with obesity.8 In the current review, we will discuss the role of the l-arginine–NO pathway in the long-term regulation of blood pressure, its complex inter-relationship with other key neurohormonal and biochemical determinants of arterial pressure, and the way in which this system becomes deranged in obesity-related hypertension. NO is well recognized as a pivotal endogenous modulator of vascular tone and endothelial function.9 l-Arginine is the sole substrate for NO formation and transport of extracellular l-arginine via cationic amino acid transporter-1 (CAT1) is a rate limiting factor for endothelial NO synthase (eNOS)–dependent NO formation (Figure 1).9–11 This occurs despite the fact that intracellular concentration of l-arginine far exceeds the Michaelis constant Km of eNOS for l-arginine, and this phenomenon is commonly referred to as the l-arginine paradox.9 The precise factors underlying this paradox remain to be determined, but …

Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l-arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l-arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l-arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l-arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1nM insulin+1mM l-arginine exhibited significant (all P

New FindingsWhat is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy?What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required.Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n=12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n=12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P=0.05). Cardiac collagen typeI mRNA expression was greater in WT mice with TAC (by 22%; P=0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in -myosin heavy chain mRNA expression in CAT+ mice compared with WT (P0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Mechanisms underlying the renal pathology in cardiorenal syndrome (CRS) type 2 remain elusive. We hypothesised that renal glutathione deficiency is central to the development of CRS type 2. Glutathione precursor, N-acetylcysteine (NAC;40 mg/kg/day; 8 weeks) or saline were administered to transgenic mice with dilated cardiomyopathy (DCM) and wild-type (WT) controls. Cardiac structure, function and glutathione levels were assessed at the end of this protocol. Renal fibrosis, glutathione content, expression of inflammatory and fibrotic markers, and function were also evaluated. In both genotypes, NAC had minimal effect on cardiac glutathione, structure and function (P ≥ 0.20). In NAC treated DCM mice, loss of glomerular filtration rate (GFR), tubulointerstitial and glomerular fibrosis and renal oxidised glutathione levels were attenuated by 38%, 99%, 70% and 52% respectively, compared to saline treated DCM mice (P ≤ 0.01). Renal expression of PAI-1 was greater in saline treated DCM mice than in WT mice (P P = 0.03). Renal IL-10 expression was greater in the former cohort compared to the latter (P

Observational studies indicate that psychological stress may contribute to the pathogenesis of hypertension and this may be further accentuated by factors such as endothelial dysfunction. On this basis, we aimed to determine whether oxidative stress enhances pressor responses to stressful stimuli and whether augmenting endothelial function by increasing the transport of L-arginine can counter the effects of oxidative stress. Telemetry probes were used to measure mean arterial pressure (MAP) in wild-type (WT; n = 6) and endothelial cationic amino acid transporter-1 (CAT-1)-overexpressing (CAT+) mice (n = 6) before and during an aversive (restraint) and non-aversive (almond feeding) stressor. The superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg/kg per day; 14 days) was then administered via a minipump to induce oxidative stress. Stress responses to feeding and restraint were repeated during Days 11-12 of DETCA infusion. In WT mice, pressor responses to restraint and feeding were augmented during infusion of DETCA (35 ± 1 and 28 ± 1 mmHg, respectively) compared with respective pretreatment responses (28 ± 2 and 24 ± 1 mmHg, respectively; P ≤ 0.01). In CAT+ mice, pressor responses to feeding were blunted during DETCA (20 ± 1 mmHg) compared with the control response (23 ± 1 mmHg; P = 0.03). In these mice, pressor responses to restraint were similar before (28 ± 1 mmHg) and during (26 ± 1 mmHg) DETCA infusion (P = 0.26). We conclude that endothelial CAT-1 overexpression can counter the ability of oxidative stress to augment pressor responses to behavioural stress.

Endothelin is one of the most potent peptide vasoconstrictors thus far characterised. It is produced by the cleavage of its precursor big endothelin-1 by endothelin-converting enzyme-1 (ECE-1). The endothelin system which includes endothelin-1 (ET-1), ET receptors and ECE-1 is well characterised in the kidney and is known to play a key role in the pathogenesis of end-stage renal disease (ESRD). Therefore, inhibition of ECE-1 and antagonism of ET receptors represent potential therapeutic approaches for the treatment of ESRD. Here, we review the current literature on the localisation of ECE-1 in the normal kidney and how ECE-1 expression is altered in pathological conditions leading to ESRD. We also discuss the roles of neutral endopeptidase (NEP) and chymase in mediating the production of ET-1 in the kidney in ESRD. As such, we also discuss that complete inhibition of ET-1 production in the kidney requires the inhibition of ECE-1, NEP and chymase.

Purpose of reviewL-Arginine (L-Arg) is the substrate for nitric oxide (NO) formation. Reduced NO bioavailability, particularly within the renal circulation, has been identified as a key factor in the pathogenesis of hypertension. This review focuses on the pathogenic role of abnormal L-Arg transport, particularly within the kidney, in hypertension.Recent findingsMost recent studies have attempted to restore NO bioavailability in cardiovascular diseases with the use of antioxidants to reduce NO inactivation, but this approach has failed to provide beneficial effects in the clinical setting. We argue that this may be due to reduced NO formation in hypertension, which has largely been overlooked as a means of restoring NO bioavailability in cardiovascular diseases. Recent data indicate that renal L-Arg transport plays an important role in regulating both renal perfusion and function and the long-term set point of arterial pressure in health. Perturbations in the renal L-Arg transport system can give rise to abnormal renal perfusion and function, initiating hypertension and related renal damage.SummaryAccordingly, we propose that L-Arg transporters are a new treatment target in hypertension and in disease states where renal NO bioavailability is disturbed.

Alzheimer's disease is a debilitating neurological disease placing significant burden on health care budgets around the world. It is widely believed that accumulation of amyloid-beta (Aβ) in the brain is a key event that initiates neurodegeneration, thus the clearance of Aβ from brain could be a key therapeutic strategy. Aβ exists in an equilibrium in healthy individuals, and recent research would suggest that dysfunction in the clearance pathways is the driving force behind its accumulation. One mechanism of clearance is proteolytic degradation by enzymes, and increasing the expression of these enzymes in animal models of Alzheimer's disease has indeed shown promising results. This approach could be challenging to translate into the clinic given the likely need for genetic manipulation. We hypothesize that stimulating the activity of these enzymes (as opposed to increasing expression) through pharmacological agents will enhance degradation or at least prevent amyloid deposition, and is therefore another potentially novel avenue to manipulate Aβ levels for therapeutic purposes. We discuss the recent research supporting this hypothesis as well as possible drawbacks to this approach.

Endothelin‐1 (ET‐1) and nitric oxide (NO) are two highly potent vasoactive molecules with opposing effects on the vasculature. Endothelin‐converting enzyme (ECE) and nitric oxide synthase (NOS) catalyse the production of ET‐1 and NO, respectively. It is well established that these molecules play a crucial role in the initiation and progression of cardiovascular diseases and have therefore become targets of therapy. Many studies have examined the mechanism(s) by which NO regulates ET‐1 production. Expression and localization of ECE‐1 is a key factor that determines the rate of ET‐1 production. ECE‐1 can either be membrane bound or be released from the cell surface to produce a soluble form. NO has been shown to reduce the expression of both membrane‐bound and soluble ECE‐1. Several studies have examined the mechanism(s) behind NO‐mediated inhibition of ECE expression on the cell membrane. However, the precise mechanism(s) behind NO‐mediated inhibition of soluble ECE production are unknown. We hypothesize that both exogenous and endogenous NO, inhibits the production of soluble ECE‐1 by preventing its release via extracellular vesicles (e.g., exosomes), and/or by inhibiting the activity of A Disintegrin and Metalloprotease‐17 (ADAM17). If this hypothesis is proven correct in future studies, these pathways represent targets for the therapeutic manipulation of soluble ECE‐1 production.

We read the review article entitled “Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition” with great interest.1 As alluded to by the authors themselves, one of the potential concerns of this therapy relates to reduced amyloid-β plaque degradation resulting from neprilysin inhibition. Amyloid-β deposition in the brain plays a central role in the pathogenesis of Alzheimer disease. …

Background: Dietary intake of fruit and vegetables is associated with lower incidence of hypertension, but the mechanisms involved have not been elucidated. Here, we evaluated the effect of a high-fiber diet and supplementation with the short-chain fatty acid acetate on the gut microbiota and the prevention of cardiovascular disease. Methods: Gut microbiome, cardiorenal structure/function, and blood pressure were examined in sham and mineralocorticoid excess–treated mice with a control diet, high-fiber diet, or acetate supplementation. We also determined the renal and cardiac transcriptome of mice treated with the different diets. Results: We found that high consumption of fiber modified the gut microbiota populations and increased the abundance of acetate-producing bacteria independently of mineralocorticoid excess. Both fiber and acetate decreased gut dysbiosis, measured by the ratio of Firmicutes to Bacteroidetes, and increased the prevalence of Bacteroides acidifaciens . Compared with mineralocorticoid-excess mice fed a control diet, both high-fiber diet and acetate supplementation significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and left ventricular hypertrophy. Acetate had similar effects and markedly reduced renal fibrosis. Transcriptome analyses showed that the protective effects of high fiber and acetate were accompanied by the downregulation of cardiac and renal Egr1 , a master cardiovascular regulator involved in cardiac hypertrophy, cardiorenal fibrosis, and inflammation. We also observed the upregulation of a network of genes involved in circadian rhythm in both tissues and downregulation of the renin-angiotensin system in the kidney and mitogen-activated protein kinase signaling in the heart. Conclusions: A diet high in fiber led to changes in the gut microbiota that played a protective role in the development of cardiovascular disease. The favorable effects of fiber may be explained by the generation and distribution of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate. Acetate effected several molecular changes associated with improved cardiovascular health and function.

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease.

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 μmol·kg−1·min−1; 30 min) and subsequent superimposition of l-arginine (100 μmol·kg−1·min−1; 30 min), the NO synthase inhibitor NG-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 μg·kg−1·min−1) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (−15 ± 7%; P = 0.07) and reduced medullary NO concentration (−9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[3H]arginine transport was less in SHR compared with SD rats ( P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.

Aim: To examine whether reduced renal arginine transport increases the responsiveness of the renal circulation to angiotensin II in salt sensitivity, renal perfusion responses to angiotensin II were examined in the presence of l-arginine transport inhibitor, l-lysine and subsequent l-arginine in Sprague Dawley (SD) and Dahl salt-sensitive (Dahl S) rats. Methods: Laser Doppler probes and a transonic flow probe were used to measure regional renal perfusion and total renal perfusion respectively. Renal perfusion responses to intravenous (i.v.) angiotensin II were sequentially examined under control conditions and during i.v. infusion of l-lysine, l-arginine or nitric oxide synthase inhibitor, NG-nitro-l-arginine. Results: Angiotensin II (10 and 100 ng kg−1 min−1, i.v.) reduced total renal (−10 ± 3 and −36 ± 5%) and cortical (−10 ± 2 and −28 ± 4%) but not medullary perfusion in SD rats. In these rats l-lysine enhanced the renal perfusion response (P = 0.003), whereas subsequent l-arginine reversed this effect (P = 0.04). Angiotensin II reduced total renal, cortical and medullary perfusion in Dahl S rats. In Dahl S rats fed high salt, l-lysine did not affect renal perfusion responses to angiotensin II, but subsequent l-arginine blunted the renal blood flow response (P = 0.01) and increased the medullary perfusion during angiotensin II infusion (P = 0.006). Conclusion: Intact renal l-arginine transport attenuates the vasoconstrictor effects of circulating angiotensin II in the renal cortex in SD rats. l-arginine also plays an important role in protecting the renal medullary circulation from the ischemic effects of angiotensin II in Dahl S rats.

1. l-Arginine is the substrate for vascular nitric oxide (NO) formation. Under normal physiological conditions, intracellular l-arginine levels far exceed the K(m) of NO synthase for l-arginine. However, endogenous NO formation is dependent on extracellular l-arginine concentrations, giving rise to the concept of the 'l-arginine paradox'. 2. Nitric oxide production in epithelial and endothelial cells is closely coupled to cellular l-arginine uptake, indicating that l-arginine transport mechanisms play a major role in the regulation of NO-dependent function. 3. Consistent with the data in endothelial and epithelial cells are functional data indicating that exogenous l-arginine can increase renal vascular and tubular NO bioavailability and thereby influence kidney perfusion, function and arterial pressure. The integrated effect of increased cellular l-arginine transport is to lower arterial pressure. Therefore, the use of l-arginine in the treatment of hypertension warrants investigation. 4. Low NO bioavailability is central to the development and maintenance of hypertension and to related endothelial dysfunction and target organ damage. We propose that l-arginine can interrupt the vicious cycle that initiates and maintains low NO in hypertension by increasing the formation of NO.

A highly complex interplay exists between the heart and kidney in the setting of both normal and abnormal physiology. In the context of heart failure, a pathophysiological condition termed the cardiorenal syndrome (CRS) exists whereby dysfunction in the heart or kidney can accelerate pathology in the other organ. The mechanisms that underpin CRS are complex, and include neuro-hormonal activation, oxidative stress and endothelial dysfunction. The endothelium plays a central role in the regulation of both cardiac and renal function, and as such impairments in endothelial function can lead to dysfunction of both these organs. In particular, reduced bioavailability of nitric oxide (NO) is a key pathophysiologic component of endothelial dysfunction. The synthesis of NO by the endothelium is critically dependent on the plasmalemmal transport of its substrate, L-arginine, via the cationic amino acid transporter-1 (CAT1). Impaired L-arginine-NO pathway activity has been demonstrated individually in heart and renal failure. Recent findings suggest abnormalities of the L-arginine-NO pathway also play a role in the pathogenesis of CRS and thus this pathway may represent a potential new target for the treatment of heart and renal failure.

We examined the roles of cyclooxygenase products and of interactions between the cyclooxygenase and nitric oxide systems in the mechanisms underlying the relative insensitivity of medullary perfusion to renal nerve stimulation (RNS) in anaesthetized rabbits. To this end we examined the effects of ibuprofen and N(G)-nitro-L: -arginine (L-NNA), both alone and in combination, on the responses of regional kidney perfusion to RNS. Under control conditions, RNS produced frequency-dependent reductions in total renal blood flow (RBF; -82+/-3% at 6 Hz), cortical laser-Doppler flux (CLDF; -84+/-4% at 6 Hz) and, to a lesser extent, medullary laser-Doppler flux (MLDF; -46+/-7% at 6 Hz). Ibuprofen did not affect these responses significantly, suggesting that cyclooxygenase products have little net role in modulating renal vascular responses to RNS. L-NNA enhanced RBF (P=0.002), CLDF (P=0.03) and MLDF (P=0.03) responses to RNS. As we have shown previously, this effect of L-NNA was particularly prominent for MLDF at RNS frequencies < or = 1.5 Hz. Subsequent administration of ibuprofen, in L-NNA-pretreated rabbits, did not affect responses to RNS significantly. We conclude that counter-regulatory actions of NO, but not of prostaglandins, partly underlie the relative insensitivity of medullary perfusion to renal nerve activation.

This study examined the effects of renal arterial infusion of a selective cytochrome P-450 epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 2 mg/kg plus 1.5 mg·kg−1·h−1), on renal hemodynamic responses to infusions of [Phe2,Ile3,Orn8]vasopressin and ANG II into the renal artery of anesthetized rabbits. MS-PPOH did not affect basal renal blood flow (RBF) or cortical or medullary blood flow measured by laser-Doppler flowmetry (CLDF/MLDF). In vehicle-treated rabbits, [Phe2,Ile3,Orn8]vasopressin (30 ng·kg−1·min−1) reduced MLDF by 62 ± 7% but CLDF and RBF were unaltered. In MS-PPOH-treated rabbits, RBF and CLDF fell by 51 ± 8 and 59 ± 13%, respectively, when [Phe2,Ile3,Orn8]vasopressin was infused. MS-PPOH had no significant effects on the MLDF response to [Phe2,Ile3,Orn8]vasopressin (43 ± 9% reduction). ANG II (20 ng·kg−1·min−1) reduced RBF by 45 ± 10% and CLDF by 41 ± 14%, but MLDF was not significantly altered. MS-PPOH did not affect blood flow responses to ANG II. Formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DiHETEs) was 49% lower in homogenates prepared from the renal cortex of MS-PPOH-treated rabbits than from vehicle-treated rabbits. MS-PPOH had no effect on the renal formation of 20-hydroxyeicosatetraenoic acid (20-HETE). Incubation of renal cortical homogenates from untreated rabbits with [Phe2,Ile3,Orn8]vasopressin (0.2–20 ng/ml) did not affect formation of EETs, DiHETEs, or 20-HETE. These results do not support a role for de novo EET synthesis in modulating renal hemodynamic responses to ANG II. However, EETs appear to selectively oppose V1-receptor-mediated vasoconstriction in the renal cortex but not in the medullary circulation and contribute to the relative insensitivity of medullary blood flow to V1-receptor activation.

What is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy? What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required. Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15 weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n = 12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n = 12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P ≤ 0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P = 0.05). Cardiac collagen type I mRNA expression was greater in WT mice with TAC (by 22%; P = 0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in β-myosin heavy chain mRNA expression in CAT+ mice compared with WT (P ≤ 0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P ≤ 0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Augmenting endothelial specific transport of the nitric oxide precursor L-arginine via cationic amino acid transporter-1 (CAT1) can prevent obesity related hypertension. We tested the hypotheses that CAT1 overexpression prevents obesity-induced hypertension by buffering the influence of the sympathetic nervous system (SNS) on the maintenance of arterial pressure and by buffering pressor responses to stress. Wild type (WT; n=13) and CAT1 overexpressing mice (CAT+; n=13) were fed a normal or a high fat diet for 20 weeks. Mice fed a high fat diet were returned to the control diet before experiments commenced. Baseline mean arterial pressure (MAP) and effects of restraint-, shaker- and almond feeding-stress and ganglionic blockade (pentolinium; 5 mg/kg; i.p.) on MAP were determined in conscious mice. Fat feeding increased body weight to a similar extent in WT and CAT+ but MAP was greater only in WT compared to appropriate controls (by 29%). The depressor response to pentolinium was 65% greater in obese WT than lean WT (P < 0.001), but was similar in obese and lean CAT+ (P = 0.65). In lean WT and CAT+, pressor responses to shaker and feeding stress, but not restraint stress, were less in the latter genotype compared to the former (P ≤ 0.001). Pressor responses to shaker and feeding stress were less in obese WT than lean WT (P ≤ 0.001), but similar in obese and lean CAT+. The increase in MAP in response to restraint stress was less in obese WT (22 ± 2%), but greater in obese CAT+ (37 ± 2%), when compared to respective lean WT (31 ± 3%) and lean CAT+ controls (27 ± 2%; P ≤ 0.02). We conclude that CAT1 overexpression prevents obesity-induced hypertension by reducing the influence of the SNS on the maintenance of arterial pressure but not by buffering pressor responses to stress.

We tested for regional differences in perfusion responses, within the renal medulla and cortex, to renal nerve stimulation in pentobarbital sodium-anesthetized rabbits. Laser-Doppler flux (LDF) was monitored at various depths below the cortical surface (1-15 mm). Basal cortical LDF (1-3 mm, approximately 200-450 U) was greater than medullary LDF (5-15 mm, approximately 70-160 U), but there were no statistically significant differences in basal LDF within these regions. The background LDF signal during aortic occlusion was similar in the cortex (2 mm, 31 U) and outer medulla (7 mm, 31 U), but slightly greater in the inner medulla (12 mm, 44 U). During electrical stimulation of the renal nerves (0.5-8 Hz), cortical LDF and total renal blood flow were similarly progressively reduced with increasing stimulus frequency. Medullary LDF (measured between 5 and 15 mm) was overall less responsive than cortical LDF. For example, 4-Hz stimulation reduced inner medullary LDF (9 mm) by 19 +/- 6% but reduced cortical LDF (1 mm) by 54 +/- 11%. However, medullary LDF responses to nerve stimulation were similar at all depths measured. Our results indicate that while the vascular elements controlling medullary perfusion are less sensitive to the effects of electrical stimulation of the renal nerves than are those controlling cortical perfusion, sensitivity within these vascular territories appears to be relatively homogeneous.

To determine whether differential release of nitric oxide underlies the diversity of regional kidney blood flow responses to vasoactive agents, this study examined how nitric oxide synthase blockade with IV N(G)-nitro-L-arginine (L-NNA), and also IV L-NNA plus co-infusion of glyceryl trinitrate, affected responses to renal arterial boluses and infusions of vasoactive agents. L-NNA, but not vehicle, or L-NNA plus glyceryl trinitrate, increased mean arterial pressure (35%) and reduced renal blood flow (20%), cortical perfusion (11%), and medullary perfusion (54%). L-NNA plus glyceryl trinitrate, but not L-NNA alone, blunted renal vasodilatation in response to boluses of bradykinin and acetylcholine, abolished increased medullary perfusion after bolus angiotensin II, and enhanced reductions in medullary perfusion, and to a lesser extent those in renal blood flow and cortical perfusion, during norepinephrine infusion. Neither L-NNA, nor L-NNA plus glyceryl trinitrate, affected responses to infusions of angiotensin II, [Phe(2),Ile(3),Orn(8)]-vasopressin, or endothelin-1. The data indicate roles for nitric oxide in angiotensin II-induced increases in medullary perfusion and in protecting medullary perfusion from norepinephrine-induced vasoconstriction. However, differential engagement of nitric oxide synthase cannot completely account for the diversity of responses of regional kidney perfusion to vasoactive agents. Effects of nitric oxide synthase blockade on renal vascular responses to vasoactive agents were revealed only when glyceryl trinitrate was co-infused to restore resting nitrergic vasodilator tone. This may reflect interactions between nitric oxide and other vasodilator mediators, in modulating renal hemodynamic responses to vasoactive agents.

AimHypertension is a major clinical complication of obesity. Our previous studies show that abnormal uptake of the nitric oxide precursor L-arginine, via the cationic amino acid transporter-1 (CAT1), contributes to endothelial dysfunction in cardiovascular disease. In this study, we tested the hypothesis that abnormal L-arginine transport may be a key mediator of obesity-induced hypertension.MethodsMean arterial pressure (MAP) was monitored by telemetry in conscious wild-type (WT; n=13) mice, and transgenic mice with endothelial-specific overexpression of CAT1 (CAT+; n=14) fed a normal or a high fat diet for 20weeks. Renal angiotensin II (Ang II), CAT1 mRNA and plasma nitrate/nitrite levels were then quantified. In conjunction, plasma nitrate/nitrite levels were assessed in obese normotensive (n=15) and obese hypertensive subjects (n=15).ResultsBoth genotypes of mice developed obesity when fed a high fat diet (P0.002). Fat fed WT mice had 13% greater MAP and 78% greater renal Ang II content, 42% lesser renal CAT1 mRNA levels and 42% lesser plasma nitrate/nitrite levels, than WT mice fed a normal fat diet (P0.02). In contrast, none of these variables were significantly altered by high fat feeding in CAT+ mice (P0.36). Plasma nitrate/nitrite levels were 17% less in obese hypertensives compared with obese normotensives (P=0.02).ConclusionCollectively, these data indicate that obesity-induced down-regulation of CAT1 expression and subsequent reduced bioavailability of nitric oxide may contribute to the development of obesity-induced hypertension.

Tissue hypoxia has been demonstrated, in both the renal cortex and medulla, during the acute phase of reperfusion after ischemia induced by occlusion of the aorta upstream from the kidney. However, there are also recent clinical observations indicating relatively well preserved oxygenation in the nonfunctional transplanted kidney. To test whether severe acute kidney injury can occur in the absence of widespread renal tissue hypoxia, we measured cortical and inner medullary tissue Po2 as well as total renal O2 delivery (Do2) and O2 consumption (V̇o2) during the first 2 h of reperfusion after 60 min of occlusion of the renal artery in anesthetized rats. To perform this experiment, we used a new method for measuring kidney Do2 and V̇o2 that relies on implantation of fluorescence optodes in the femoral artery and renal vein. We were unable to detect reductions in renal cortical or inner medullary tissue Po2 during reperfusion after ischemia localized to the kidney. This is likely explained by the observation that V̇o2 (−57%) was reduced by at least as much as Do2 (−45%), due to a large reduction in glomerular filtration (−94%). However, localized tissue hypoxia, as evidence by pimonidazole adduct immunohistochemistry, was detected in kidneys subjected to ischemia and reperfusion, particularly in, but not exclusive to, the outer medulla. Thus, cellular hypoxia, particularly in the outer medulla, may still be present during reperfusion even when reductions in tissue Po2 are not detected in the cortex or inner medulla.

Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 andmore » 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.« less

This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 µM), NOS substrate L-Arg (200-1,000 µM), a L-Arg transport inhibitor (L-Lys; 10 µM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 µM). The effect of L-Arg (1,000 µM) was also tested in the presence of L-Lys (10 µM), L-Gln (100 µM) and L-NAME (10-100 µM). Ultracentrifugation (100,000×g, 4 °C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P < 0.05, unpaired t test, n = 4) in the presence of DETA (75.31 ± 3.59; 800 µM) and L-Arg (60.97 ± 9.22; 1,000 µM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 µM; 99.19 ± 0.58) and L-Gln (100 µM; 104.41 ± 0.65). In addition, the presence of L-Lys (10 µM) significantly blocked the L-Arg (1,000 µM)-induced reduction in soluble ECE-1 levels (122.38 ± 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.

Aim: Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial L-arginine transporter, cationic amino acid transporter-1 (CAT1), can prevent oxidative stress-induced hypertension. Methods: Wild-type mice (WT; n = 9) and endothelial CAT1 overexpressing (CAT+ )m ice (n = 6) had telemetry probes implanted for the measurement of mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg kg 1 day 1 ; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2-isoprostane levels. Results: Basal MAP was less in CAT+ (92 2 mmHg; n = 6) than in WT (98 2 mmHg; n = 9; P< 0.001). During DETCA infusion, MAP was increased in WT (by 4.2 0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls (PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2-isoprostane levels (by 67 11%; P = 0.05) in WT but not in CAT+. Total renal F2-isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. Conclusion: Augmented endothelial L-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress.

Obesity related hypertension is a major risk factor for resistant hypertension. We do not completely understand the mechanism(s) underlying the development of obesity related hypertension which hinders the development of novel treatment strategies for this condition. Data from experimental studies and small clinical trials indicate that transport of l-arginine, the substrate for nitric oxide (NO), and subsequent NO production are reduced in obesity induced hypertension. Reduced NO bioavailability can induce hypertension via multiple mechanisms. Mirmiran et al. recently analyzed data from a large population study and found that the association between dietary l-arginine and serum nitrate and nitrite was weakened in obese hypertensive subjects compared to obese normotensives. These data suggest that l-arginine dependent NO production is impaired in the former group compared to the latter which may represent a novel mechanism contributing to hypertension in the setting of obesity.

1. Angiotensin (Ang) II has multiple actions in the renal medullary circulation. It can induce vasodilatation and blunt the response of medullary blood flow (MBF) to renal nerve activation through AT(1) receptor-mediated release of nitric oxide (NO) and/or vasodilator prostaglandins. These actions require high intravascular and/or intratubular AngII concentrations, so are not apparent under physiological conditions. 2. Nevertheless, these mechanisms blunt the responsiveness of MBF to AT(1) receptor-mediated vasoconstriction. When these protective mechanisms fail, as when oxidative stress reduces NO bioavailability in the medullary circulation, AngII reduces MBF. If sustained, reduced MBF leads to the development of hypertension. 3. Chronic activation of the renin-angiotensin system (RAS) induces oxidative stress in the kidney. Therefore, MBF may be reduced in models of hypertension associated with RAS activation both because AngII levels per se are increased and because of increased responsiveness of MBF to AngII-induced vasoconstriction. 4. Endogenous AngII enhances the responsiveness of MBF to renal nerve stimulation, whereas NO blunts it. Chronic RAS activation and/or oxidative stress should therefore be expected to enhance MBF responses to renal nerve stimulation. Consistent with this, reductions in MBF induced by renal nerve stimulation are enhanced in rabbits with AngII-induced hypertension, renovascular hypertension or after 9 weeks of fat feeding. 5. We conclude that the ability of endogenous AngII to reduce MBF and enhance the response of MBF to activation of the renal nerves could contribute to the development of hypertension under conditions of RAS activation, especially if accompanied by increased renal sympathetic nerve activity.

We tested the hypothesis that activation of angiotensin type 2 (AT2) receptors, by both exogenous and endogenous ANG II, modulates neurally mediated vasoconstriction in the renal cortical and medullary circulations. Under control conditions in pentobarbital-anesthetized rabbits, electrical stimulation of the renal nerves (RNS; 0.5–8 Hz) reduced renal blood flow (RBF; −88 ± 3% at 8 Hz) and cortical perfusion (CBF; −92 ± 2% at 8 Hz) more than medullary perfusion (MBF; −67 ± 6% at 8 Hz). Renal arterial infusion of ANG II, at a dose titrated to reduce RBF by ∼40–50% (5–50 ng·kg−1·min−1) blunted responses of MBF to RNS, without significantly affecting responses of RBF or CBF. Subsequent administration of PD123319 (1 mg/kg plus 1 mg·kg−1·h−1) during continued renal arterial infusion of ANG II did not significantly affect responses of RBF or CBF to RNS but enhanced responses of MBF, so that they were similar to those observed under control conditions. In contrast, administration of PD123319 alone blunted responses of CBF and MBF to RNS. Subsequent renal arterial infusion of ANG II in PD123319 -pretreated rabbits restored CBF responses to RNS back to control levels. In contrast, ANG II infusion in PD123319 -pretreated rabbits did not alter MBF responses to RNS. These data indicate that exogenous ANG II can blunt neurally mediated vasoconstriction in the medullary circulation through activation of AT2 receptors. However, AT2-receptor activation by endogenous ANG II appears to enhance neurally mediated vasoconstriction in both the cortical and medullary circulations.

We investigated the roles of the renin-angiotensin system and the significance of interactions between angiotensin II and nitric oxide, in responses of regional kidney perfusion to electrical renal nerve stimulation (RNS) in pentobarbital sodium-anesthetized rabbits. Under control conditions, RNS (0.5–8 Hz) reduced total renal blood flow (RBF; −89 ± 3% at 8 Hz) and cortical perfusion (CBF; −90 ± 2% at 8 Hz) more than medullary perfusion (MBF; −55 ± 5% at 8 Hz). Angiotensin II type 1 (AT1)-receptor antagonism (candesartan) blunted RNS-induced reductions in RBF ( P = 0.03), CBF ( P = 0.007), and MBF ( P = 0.04), particularly at 4 and 8 Hz. Nitric oxide synthase inhibition with NG-nitro-l-arginine (l-NNA) enhanced RBF ( P = 0.003), CBF ( P = 0.001), and MBF ( P = 0.03) responses to RNS, particularly at frequencies of 2 Hz and less. After candesartan pretreatment, l-NNA significantly enhanced RNS-induced reductions in RBF ( P = 0.04) and CBF ( P = 0.007) but not MBF ( P = 0.66). Renal arterial infusion of angiotensin II (5 ng·kg−1·min−1) selectively enhanced responses of MBF to RNS in l-NNA-pretreated but not in vehicle-pretreated rabbits. In contrast, greater doses of angiotensin II (5–15 ng·kg−1·min−1) blunted responses of MBF to RNS in rabbits with intact nitric oxide synthase. These results suggest that endogenous angiotensin II enhances, whereas nitric oxide blunts, neurally mediated vasoconstriction in the renal cortical and medullary circulations. In the renal medulla, but not the cortex, angiotensin II also appears to be able to blunt neurally mediated vasoconstriction.

1. The aim of the present study was to examine the roles of cyclo-oxygenase (COX)- and lipoxygenase (LOX)-dependent arachidonate signalling cascades in the control of regional kidney blood flow. 2. In pentobarbitone-anaesthetized rabbits treated with NG-nitro-l-arginine and glyceryl trinitrate to 'clamp' nitric oxide, we determined the effects of ibuprofen (a COX inhibitor) and esculetin (a LOX inhibitor) on resting systemic and renal haemodynamics and responses to renal arterial infusions of vasoconstrictors. 3. Ibuprofen increased mean arterial pressure (14 +/- 5%) and reduced medullary laser Doppler flux (MLDF; 26 +/- 6%) when administered with esculetin. A similar pattern of responses was observed when ibuprofen was given alone, although the reduction in MLDF was not statistically significant. Esculetin tended to increase renal blood flow (RBF; 16 +/- 7%) and MLDF (28 +/- 13%) when given alone, but not when combined with ibuprofen. 4. After vehicle, renal arterial infusions of noradrenaline, angiotensin II and endothelin-1 reduced RBF and cortical laser Doppler flux (CLDF), but not MLDF. In contrast, renal arterial [Phe2,Ile3,Orn8]-vasopressin reduced MLDF but not RBF or CLDF. Ibuprofen alone did not significantly affect these responses. Esculetin, when given alone, but not when combined with ibuprofen, enhanced noradrenaline-induced renal vasoconstriction. In contrast, esculetin did not significantly affect responses to [Phe2,Ile3,Orn8]-vasopressin, angiotensin II or endothelin-1. 5. We conclude that COX products contribute to the maintenance of arterial pressure and renal medullary perfusion under 'nitric oxide clamp' conditions, but not to renal haemodynamic responses to the vasoconstrictors we tested. Lipoxygenase products may blunt noradrenaline-induced vasoconstriction, but our observations may, instead, reflect LOX-independent effects of esculetin.

Summary 1. To determine whether differential release of products of arachidonic acid metabolism, via the cyclo-oxygenase pathway, underlies the diversity of responses of regional kidney perfusion to vasoactive agents, we tested the effects of intravenous indomethacin on responses to renal arterial bolus doses of vasoactive agents in pentobarbitone-anaesthetized rabbits. 2. Total renal blood flow (RBF) and regional kidney perfusion were determined by transit time ultrasound flowmetry and laser-Doppler flowmetry, respectively. 3. Responses of regional kidney blood flow to vasoactive agents were diverse: noradrenaline reduced cortical but not medullary perfusion, [Phe2,Ile3,Orn8]-vasopressin reduced medullary perfusion more than cortical perfusion, endothelin-1 and angiotensin II increased medullary perfusion in the face of reduced cortical perfusion, while acetylcholine, bradykinin and the nitric oxide donor methylamine hexamethylene methylamine (MAHMA) NONOate all increased both cortical and medullary perfusion. 4. Indomethacin administration was followed by reductions in total RBF (17 ± 6%), cortical perfusion (13 ± 5%) and medullary perfusion (40 ± 8%). Angiotensin II- and endothelin-1-induced increases in medullary perfusion were abolished by indomethacin, but indomethacin had no significant effects on responses of regional kidney perfusion to acetylcholine, bradykinin, MAHMA NONOate, noradrenaline and [Phe2,Ile3,Orn8]-vasopressin. 5. Our results suggest that vasodilator cyclo-oxygenase products contribute to the maintenance of resting renal vascular tone, particularly in vascular elements controlling medullary perfusion. Cyclo-oxygenase products also appear to mediate endothelin-1- and angiotensin II-induced increases in medullary perfusion. However, regionally specific engagement of cyclo-oxygenase-dependent arachidonic acid metabolism does not appear to contribute to the differential effects of noradrenaline and [Phe2,Ile3,Orn8]-vasopressin on cortical and medullary perfusion.

O ST E R A B ST R A C T S vs. -17.23 2.23%, p1⁄4