Adenosine A2A and A3 Receptors as Targets for the Treatment of Hypertensive-Diabetic Nephropathy.

Diabetic nephropathy (DN) and hypertension are prime causes for end-stage renal disease (ESRD) that often coexist in patients, but are seldom studied in combination. Kidney adenosine levels are markedly increased in diabetes, and the expression and function of renal adenosine receptors are altered in experimental diabetes. The aim of this work is to explore the impact of endogenous and exogenous adenosine on the expression/distribution profile of its receptors along the nephron of hypertensive rats with experimentally-induced diabetes. Using spontaneously hypertensive (SHR) rats rendered diabetic with streptozotocin (STZ), we show that treatment of SHR-STZ rats with an agonist of adenosine receptors increases A2A immunoreactivity in superficial glomeruli (SG), proximal tubule (PCT), and distal tubule (DCT). Differently, treatment of SHR-STZ rats with a xanthinic antagonist of adenosine receptors decreases adenosine A3 immunoreactivity in SG, PCT, DCT, and collecting duct. There is no difference in the immunoreactivity against the adenosine A1 and A2B receptors between the experimental groups. The agonist of adenosine receptors ameliorates renal fibrosis, probably via A2A receptors, while the antagonist exacerbates it, most likely due to tonic activation of A3 receptors. The reduction in adenosine A3 immunoreactivity might be due to receptor downregulation in response to prolonged activation. Altogether, these results suggest an opposite regulation exerted by endogenous and exogenous adenosine upon the expression of its A2A and A3 receptors along the nephron of hypertensive diabetic rats, which has a functional impact and should be taken into account when considering novel therapeutic targets for hypertensive-diabetic nephropathy.

Determinants of renal oxygen metabolism during low Na+ diet: effect of angiotensin II AT1 and aldosterone receptor blockade.

KEY POINTS: Reducing Na+ intake reduces the partial pressure of oxygen in the renal cortex and activates the renin-angiotensin-aldosterone system. In the absence of high blood pressure, these consequences of dietary Na+ reduction may be detrimental for the kidney. In a normotensive animal experimental model, reducing Na+ intake for 2 weeks increased renal oxygen consumption, which was normalized by mineralocorticoid receptor blockade. Furthermore, blockade of the angiotensin II AT1 receptor restored cortical partial pressure of oxygen by improving oxygen delivery. This shows that increased activity of the renin-angiotensin-aldosterone system contributes to increased oxygen metabolism in the kidney after 2 weeks of a low Na+ diet. The results provide insights into dietary Na+ restriction in the absence of high blood pressure, and its consequences for the kidney. ABSTRACT: Reduced Na+ intake reduces the PO2 (partial pressure of oxygen) in the renal cortex. Upon reduced Na+ intake, reabsorption along the nephron is adjusted with activation of the renin-angiotensin-aldosterone system (RAAS). Thus, we studied the effect of reduced Na+ intake on renal oxygen homeostasis and function in rats, and the impact of intrarenal angiotensin II AT1 receptor blockade using candesartan and mineralocorticoid receptor blockade using canrenoic acid potassium salt (CAP). Male Sprague-Dawley rats were fed standard rat chow containing normal (0.25%) and low (0.025%) Na+ for 2 weeks. The animals were anaesthetized (thiobutabarbital 120 mg kg-1 ) and surgically prepared for kidney oxygen metabolism and function studies before and after acute intrarenal arterial infusion of candesartan (4.2 µg kg-1 ) or intravenous infusion of CAP (20 mg kg-1 ). Baseline mean arterial pressure and renal blood flow were similar in both dietary groups. Fractional Na+ excretion and cortical oxygen tension were lower and renal oxygen consumption was higher in low Na+ groups. Neither candesartan nor CAP affected arterial pressure. Renal blood flow and cortical oxygen tension increased in both groups after candesartan in the low Na+ group. Fractional Na+ excretion was increased and oxygen consumption reduced in the low Na+ group after CAP. These results suggest that blockade of angiotensin II AT1 receptors has a major impact upon oxygen delivery during normal and low Na+ conditions, while aldosterone receptors mainly affect oxygen metabolism following 2 weeks of a low Na+ diet.

NADPH oxidase 1 is a novel pharmacological target for the development of an antiplatelet drug without bleeding side effects.

Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1-/- and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.

Diabetes downregulates renal adenosine A2A receptors in an experimental model of hypertension.

Studies on diabetic nephropathy rarely take into account that the co-existence of diabetes and hypertension is frequent and further aggravates the prognosis of renal dysfunction. Adenosine can activate four subtypes of adenosine receptors (A1, A2A, A2B and A3) and has been implicated in diabetic nephropathy. However, it is not known if, in hypertensive conditions, diabetes alters the presence/distribution profile of renal adenosine receptors. The aim of this work was to describe the presence/distribution profile of the four adenosine receptors in six renal structures (superficial/deep glomeruli, proximal/distal tubules, loop of Henle, collecting tubule) of the hypertensive kidney and to evaluate whether it is altered by diabetes. Immunoreactivities against the adenosine receptors were analyzed in six renal structures from spontaneously hypertensive rats (SHR, the control group) and from SHR rats with diabetes induced by streptozotocyin (SHR-STZ group). Data showed, for the first time, that all adenosine receptors were present in the kidney of SHR rats, although the distribution pattern was specific for each adenosine receptor subtype. Also, induction of diabetes in the SHR was associated with downregulation of adenosine A2A receptors, which might be relevant for the development of hypertensive diabetic nephropathy. This study highlights the adenosine A2A receptors as a potential target to explore to prevent and/or treat early diabetes-induced hyperfiltration, at least in hypertensive conditions.

Angiotensin II-induced hypertension in rats is only transiently accompanied by lower renal oxygenation.

Activation of the renin-angiotensin system may initiate chronic kidney disease. We hypothesised that renal hypoxia is a consequence of hemodynamic changes induced by angiotensin II and occurs prior to development of severe renal damage. Male Sprague-Dawley rats were infused continuously with angiotensin II (350 ng/kg/min) for 8 days. Mean arterial pressure (n = 5), cortical (n = 6) and medullary (n = 7) oxygenation (pO2) were continuously recorded by telemetry and renal tissue injury was scored. Angiotensin II increased arterial pressure gradually to 150 ± 18 mmHg. This was associated with transient reduction of oxygen levels in renal cortex (by 18 ± 2%) and medulla (by 17 ± 6%) at 10 ± 2 and 6 ± 1 hours, respectively after starting infusion. Thereafter oxygen levels normalised to pre-infusion levels and were maintained during the remainder of the infusion period. In rats receiving angiotensin II, adding losartan to drinking water (300 mg/L) only induced transient increase in renal oxygenation, despite normalisation of arterial pressure. In rats, renal hypoxia is only a transient phenomenon during initiation of angiotensin II-induced hypertension.

Cooperative Oxygen Sensing by the Kidney and Carotid Body in Blood Pressure Control.

Oxygen sensing mechanisms are vital for homeostasis and survival. When oxygen levels are too low (hypoxia), blood flow has to be increased, metabolism reduced, or a combination of both, to counteract tissue damage. These adjustments are regulated by local, humoral, or neural reflex mechanisms. The kidney and the carotid body are both directly sensitive to falls in the partial pressure of oxygen and trigger reflex adjustments and thus act as oxygen sensors. We hypothesize a cooperative oxygen sensing function by both the kidney and carotid body to ensure maintenance of whole body blood flow and tissue oxygen homeostasis. Under pathological conditions of severe or prolonged tissue hypoxia, these sensors may become continuously excessively activated and increase perfusion pressure chronically. Consequently, persistence of their activity could become a driver for the development of hypertension and cardiovascular disease. Hypoxia-mediated renal and carotid body afferent signaling triggers unrestrained activation of the renin angiotensin-aldosterone system (RAAS). Renal and carotid body mediated responses in arterial pressure appear to be synergistic as interruption of either afferent source has a summative effect of reducing blood pressure in renovascular hypertension. We discuss that this cooperative oxygen sensing system can activate/sensitize their own afferent transduction mechanisms via interactions between the RAAS, hypoxia inducible factor and erythropoiesis pathways. This joint mechanism supports our view point that the development of cardiovascular disease involves afferent nerve activation.

Acute SGLT inhibition normalizes O2 tension in the renal cortex but causes hypoxia in the renal medulla in anaesthetized control and diabetic rats.

Early stage diabetic nephropathy is characterized by glomerular hyperfiltration and reduced renal tissue Po2. Recent observations have indicated that increased tubular Na(+)-glucose linked transport (SGLT) plays a role in the development of diabetes-induced hyperfiltration. The aim of the present study was to determine how inhibition of SLGT impacts upon Po2 in the diabetic rat kidney. Diabetes was induced by streptozotocin in Sprague-Dawley rats 2 wk before experimentation. Renal hemodynamics, excretory function, and renal O2 homeostasis were measured in anesthetized control and diabetic rats during baseline and after acute SGLT inhibition using phlorizin (200 mg/kg ip). Baseline arterial pressure was similar in both groups and unaffected by SGLT inhibition. Diabetic animals displayed reduced baseline Po2 in both the cortex and medulla. SGLT inhibition improved cortical Po2 in the diabetic kidney, whereas it reduced medullary Po2 in both groups. SGLT inhibition reduced Na(+) transport efficiency [tubular Na(+) transport (TNa)/renal O2 consumption (Qo2)] in the control kidney, whereas the already reduced TNa/Qo2 in the diabetic kidney was unaffected by SGLT inhibition. In conclusion, these data demonstrate that when SGLT is inhibited, renal cortex Po2 in the diabetic rat kidney is normalized, which implies that increased proximal tubule transport contributes to the development of hypoxia in the diabetic kidney. The reduction in medullary Po2 in both control and diabetic kidneys during the inhibition of proximal Na(+) reabsorption suggests the redistribution of active Na(+) transport to less efficient nephron segments, such as the medullary thick ascending limb, which results in medullary hypoxia.

Diabetes-induced increase of renal medullary hydrogen peroxide and urinary angiotensinogen is similar in normotensive and hypertensive rats.

AIMS: Activation of renal renin-angiotensin system (RAS) and reactive oxygen species (ROS) are the main pathophysiological mechanisms associated with kidney injury both in diabetes and hypertension. However, the contribution to medullary damage when the two pathologies coexist has seldom been explored. MAIN METHODS: Diabetes was induced with streptozotocin in twelve week-old male Wistar and spontaneously hypertensive rats (SHR) rats; controls received vehicle. Three weeks later, systolic blood pressure (SBP), plasma and urinary angiotensinogen (AGT), renal oxidative stress and metabolic status were evaluated. KEY FINDINGS: SBP was higher in SHR-controls than in Wistar-controls (200±6 and 127±3mmHg, respectively) and decreased in SHR-diabetics but not in Wistar-diabetics (143±8 and 122±6mmHg, respectively). Renal medullary hydrogen peroxide (H2O2) production was similarly increased in diabetics (Wistar: 0.32±0.04 and 1.11±0.10nmol/mg protein, respectively; SHR: 0.40±0.05 and 0.90±0.14nmol/mg protein, respectively) and positively correlated with glycemia (Wistar: r=0.7166, SHR: r=0.7899, p<0.05) and urinary AGT excretion (Wistar: r=0.8333; SHR: r=0.8326, p<0.05). Cortical H2O2 production was higher in SHR-controls than in Wistar-controls (1.10±0.09 and 0.26±0.04nmol/mg protein, respectively) and diabetes induction decreased it in SHR (0.70±0.09nmol/mg protein). Diabetes increased urinary AGT excretion by more than 7-fold and decreased plasma AGT concentration by more than 1.5-fold in both strains. SIGNIFICANCE: Our results show that STZ-induced diabetes increases medullary H2O2 production and urinary AGT excretion with similar magnitude in normotensive and hypertensive animals. Reducing blood pressure attenuates hypertension-associated cortical damage but does not prevent medullary dysfunction.

Activation of adenosine receptors improves renal antioxidant status in diabetic Wistar but not SHR rats.

BACKGROUND: Diabetes and hypertension independently contribute to renal injury, and the major mechanisms involved are increased reactive oxygen species (ROS) bioavailability and renin-angiotensin system (RAS) activation. We investigated the role of adenosine in controlling ROS production and RAS activation associated with renal dysfunction in hypertension and diabetes. METHODS: Fourteen days after induction of diabetes with streptozotocin in 12-week-old male Wistar and spontaneously hypertensive (SHR) rats, animals were treated during 7 days with 2-chloroadenosine (CADO group, 5 mg/kg/d), a stable analogue of adenosine, or underwent a sham operation procedure. At the end of the study (day 21), intra-arterial systolic blood pressure (SBP) was measured, and 24-h urine and plasma samples and renal tissue were collected. RESULTS: CADO treatment decreased the plasma glucose concentration and glucose and protein excretion by more than 30% in both strains. CADO treatment decreased SBP in diabetic SHR rats (143 ± 8 versus 114 ± 4 mmHg, p < 0.05), but not in diabetic Wistar rats. The hypotensive effect of CADO was associated to a ∼70% increase in plasma angiotensinogen (AGT) concentration and a ∼50% decrease in urinary AGT excretion. CADO also caused a decrease in medullary and cortical hydrogen peroxide production of about 40%, which was associated with a proportional increase in glutathione peroxidase (GPx) activity in diabetic Wistar but not in diabetic SHR animals. CONCLUSIONS: These results suggest that activation of adenosine receptors improves renal antioxidant capacity in diabetic Wistar but not SHR rats, although it improves glucose metabolism in both strains. Furthermore, activation of adenosine receptors does not seem to be directly influencing AGT production.

Angiotensin II contributes to glomerular hyperfiltration in diabetic rats independently of adenosine type I receptors.

Increased angiotensin II (ANG II) or adenosine can potentiate each other in the regulation of renal hemodynamics and tubular function. Diabetes is characterized by hyperfiltration, yet the roles of ANG II and adenosine receptors for controlling baseline renal blood flow (RBF) or tubular Na(+) handling in diabetes is presently unknown. Accordingly, the changes in their functions were investigated in control and 2-wk streptozotocin-diabetic rats after intrarenal infusion of the ANG II AT1 receptor antagonist candesartan, the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or their combination. Compared with controls, the baseline blood pressure, RBF, and renal vascular resistance (RVR) were similar in diabetics, whereas the glomerular filtration rate (GFR) and filtration fraction (FF) were increased. Candesartan, DPCPX, or the combination increased RBF and decreased RVR similarly in all groups. In controls, the GFR was increased by DPCPX, but in diabetics, it was decreased by candesartan. The FF was decreased by candesartan and DPCPX, independently. DPCPX caused the most pronounced increase in fractional Na(+) excretion in both controls and diabetics, whereas candesartan or the combination only affected fractional Li(+) excretion in diabetics. These results suggest that RBF, via a unifying mechanism, and tubular function are under strict tonic control of both ANG II and adenosine in both control and diabetic kidneys. Furthermore, increased vascular AT1 receptor activity is a contribution to diabetes-induced hyperfiltration independent of any effect of adenosine A1 receptors.