Mónica Moreira-Rodrigues

Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice

AIMS Although toll-like receptors (TLR) are known to mediate the metabolic complications of obesity, the mechanisms underlying its activation remain largely unknown. The present study analyzed a model of diet-induced obesity in mice lacking the TLR4/TLR2 co-receptor CD14. MAIN METHODS Six-week-old male mice lacking CD14 (n = 16) were allocated to either a control diet or a high-fat high-simple carbohydrate diet (5.4 kcal/g; 35% fat; 35% sucrose), and compared with C57BL/6 (WT; n = 15) controls. After 12 weeks, body composition, basal sympathetic activity, non-invasive blood pressure and glucose tolerance were evaluated. Hepatic and adipose tissues were collected for mRNA quantification, histology and LPS incubation. KEY FINDINGS In both WT and CD14 knockout mice, obesity was accompanied by TLR2 and TLR4 upregulation. However, obese mice lacking CD14 presented decreased lipid and macrophage content in hepatic and adipose tissues, lower urinary levels of noradrenaline, decreased systolic blood pressure, reduced fasting plasma glucose and blunted glucose intolerance, compared with obese WT group. In the presence of exogenous sCD14, adipose tissue incubation with LPS-induced TLR2 and TNF-alpha upregulation in both WT and CD14 knockout obese mice. SIGNIFICANCE In our model of diet-induced obesity, mice lacking CD14 showed lower adiposity and hepatic steatosis, improved glucose homeostasis, blunted sympathetic overactivity and reduced blood pressure elevation. This was observed in the presence of preserved TLR4 and TLR2 gene expression, and intact TLR4 signaling pathways. These results suggest that CD14-mediated TLR activation might contribute to the cardiovascular and metabolic complications of obesity.

Role of Chronic Inhibition of Dopamine-Metabolizing Enzymes in the Regulation of Renal Sodium and Phosphate Excretion in the Rat Remnant Kidney

Background/Aims: The present study examined the effects of chronic selective or combined inhibition of type A monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) on daily urinary excretion of dopamine and metabolites and on natriuresis and phosphaturia in 3/4 nephrectomized (3/4nx) and Sham rats. Methods: The 3/4nx and Sham rats were placed in metabolic cages and received the MAO-A-selective inhibitor Ro-411049 (7.5 mg·kg–1 bid) and/or the COMT-selective inhibitor BIA 3-202 (30 mg·kg–1 bid) orally for 3 days during high sodium diet. Results: Selective COMT inhibition increased the urinary excretion of the deaminated metabolite (3,4-dihydroxyphenylacetic acid, DOPAC) and decreased the urinary excretion of the methylated (3-methoxytyramine, 3-MT) and deaminated plus methylated metabolite (homovanillic acid, HVA) in both groups. Selective MAO-A inhibition increased the urinary excretion of 3-MT and reduced the urinary excretion of both DOPAC and HVA in either 3/4nx or Sham rats. Combined inhibition of MAO-A and COMT did not significantly change the urinary excretion of DOPAC and markedly decreased the urinary excretion of 3-MT and HVA in both groups. Selective or combined inhibition of MAO-A and COMT did not alter the daily urinary excretion of dopamine, sodium or phosphate in either 3/4nx or Sham rats. Conclusions: Chronic selective or combined inhibition of MAO-A and COMT is not of major importance in regulating the dopamine-dependent natriuresis and phosphaturia in either 3/4nx or Sham rats.

Glycaemic control with insulin prevents the reduced renal dopamine D1 receptor expression and function in streptozotocin-induced diabetes

BACKGROUND It was demonstrated in streptozotocin (STZ)-induced diabetic rats that the D(1) receptor agonist failed to promote sodium excretion as a result of reduced renal D(1) receptor expression and decreased receptor G protein coupling. The present study examined the influence of glycaemic control with insulin on the renal D(1) receptor dysfunction in STZ-induced type 1 diabetes. METHODS Renal function, blood pressure, the natriuretic response to 5% volume expansion (VE) and the effects of the D(1) receptor agonist fenoldopam on natriuresis and on Na(+)/K(+)-ATPase activity in renal tubules were evaluated in uninephrectomized and sham-operated Wistar rats treated with STZ and compared with controls and STZ-treated rats made euglycaemic with insulin. D(1) receptor immunohistochemistry and protein abundance by western blot were also determined in all groups. RESULTS Treatment of sham and uninephrectomized rats with STZ caused a 4-fold increase in glucose plasma levels compared to controls and euglycaemic diabetic rats. A blunted natriuretic response to VE was observed in both sham and uninephrectomized hyperglycaemic diabetic rats, and this was accompanied by failure of fenoldopam to increase natriuresis and to inhibit renal Na(+)/K(+)-ATPase activity. In contrast, in both sham and uninephrectomized euglycaemic diabetic rats, the natriuretic response to VE, the fenoldopam-induced natriuresis and the accompanied inhibition of Na(+)/K(+)-ATPase activity were similar to those of the corresponding controls. D(1) receptor immunodetection and protein abundance were reduced in hyperglycaemic diabetic rats, but not in euglycaemic diabetic animals. CONCLUSIONS We conclude that the renal expression and natriuretic response to D(1) receptor activation is compromised in both sham and uninephrectomized rats with STZ-induced diabetes. These abnormalities were prevented by lowering glucose blood levels with insulin, thus providing evidence for the involvement of hyperglycaemia in the disturbances that underlie the compromised dopamine-sensitive natriuresis and increase of blood pressure in type 1 diabetes.

Concerted Action of ANP and Dopamine D1-Receptor to Regulate Sodium Homeostasis in Nephrotic Syndrome

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Local modulation of the natriuretic peptide system in the rat remnant kidney

BACKGROUND The natriuretic peptide (NP) system plays a central role in the renal adaptations to acute volume expansion. However, the modulation of the NP system in chronic renal insufficiency (CRI) remains to be elucidated. In the present study, we evaluated cardiac haemodynamics, plasma type-B natriuretic peptide (BNP) levels and the expression of natriuretic peptide receptor A (NPR-A) and NPR-C in the renal cortex (RC) and medulla (RM) of Sham and (3/4) nephrectomized ((3/4)nx) rats, up to 26 weeks after surgery. METHODS Male Wistar-Han rats (190-220 g; n = 49) were randomly assigned to (3/4)nx or Sham surgery. Two, 10 and 26 weeks after surgery, non-invasive blood pressure (BP) and left ventricular (LV) haemodynamics were performed, and urine and blood were collected for metabolic studies and plasma BNP determination. In addition, tissue samples from RC and RM were obtained for NPR-A and NPR-C quantification (RT-PCR and western blotting) as well as NPR-A immunodetection. RESULTS In (3/4)nx rats, the progressive interstitial fibrosis and tubular atrophy were accompanied by a time-dependent increase of BP and impaired natriuretic response to volume expansion (VE). This was accompanied in (3/4)nx rats by an early and time-dependent elevation of BNP circulating levels that was not associated with cardiac dysfunction or increased myocardial BNP gene expression. In (3/4)nx rats, NPR-A expression in the remnant RM was consistently reduced at 2, 10 and 26 weeks, and this was accompanied by an increase in NPR-C expression in the remnant RC from (3/4)nx rats. CONCLUSIONS BP elevation and compromised natriuretic response to VE in (3/4)nx rats is associated with increased circulating BNP levels in the absence of cardiac dysfunction. This is accompanied in (3/4)nx rats by both impaired NPR-A expression in the RM and upregulation of NPR-C in the RC suggesting a novel mechanism for NP resistance in CRI.

Renal dopaminergic system activity in rat remnant kidney up to twenty-six weeks after surgery.

AIMS In 3/4 nephrectomized (3/4nx) rats the renal dopaminergic system was suggested to be involved in the adaptive increase of sodium excretion two weeks after renal mass ablation. The aim of the present study was to evaluate the renal adaptations in sodium handling and renal dopaminergic system activity in 3/4nx rats up to twenty-six weeks after surgery. MAIN METHODS The rats were placed in metabolic cages for the collection of 24 h urine for evaluation of sodium, dopamine, dopamine precursor and metabolites. Blood pressure, aromatic L-amino acid decarboxylase (AADC) activity in proximal tubules and the effect of dopamine D(1) receptor selective antagonist (Sch-23390) on natriuresis was evaluated. KEY FINDINGS A time-dependent increase in both systolic and diastolic blood pressure was observed in 3/4nx rats, and this was accompanied by a decrease in urinary levels of dopamine and in renal AADC activity at twenty-six weeks after renal mass ablation. In contrast to what has been found two weeks after renal mass ablation, the natriuretic response to volume expansion was progressively reduced in 3/4nx rats at ten and twenty-six weeks after surgery and this was accompanied by insensitivity of natriuresis to Sch-23390. SIGNIFICANCE In conclusion the renal dopaminergic system activity is compromised in 3/4nx rats in a time-dependent manner after renal mass ablation. It is suggested that this may contribute to compromise sodium excretion and increase blood pressure, in chronic renal insufficiency.

Attenuated Aortic Vasodilation and Sympathetic Prejunctional Facilitation in Epinephrine-Deficient Mice: Selective Impairment of β2-Adrenoceptor Responses

It has been suggested that there is a link between epinephrine synthesis and the development of β2-adrenoceptor–mediated effects, but it remains to be determined whether this development is triggered by epinephrine. The aim of this study was to characterize β-adrenoceptor–mediated relaxation and facilitation of norepinephrine release in the aorta of phenylethanolamine-N-methyltransferase–knockout (Pnmt-KO) mice. Catecholamines were quantified by reverse-phase high-performance liquid chromatography–electrochemical detection. Aortic rings were mounted in a myograph to determine concentration-response curves to selective β1- or β2-adrenoceptor agonists in the absence or presence of selective β1- or β2-adrenoceptor antagonists. Aortic rings were also preincubated with [3H]norepinephrine to measure tritium overflow elicited by electrical stimulation in the presence of increasing concentrations of nonselective β- or selective β2-adrenoceptor agonists. β2-Adrenoceptor protein density was evaluated by Western blotting and β2-adrenoceptor localization by immunohistochemistry. Epinephrine is absent in Pnmt-KO mice. The potency and the maximal effect of the β2-adrenoceptor agonist terbutaline were lower in Pnmt-KO than in wild-type (WT) mice. The selective β2-adrenoceptor antagonist ICI 118,551 [(±)-erythro-(S*,S*)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride] antagonized the relaxation caused by terbutaline in WT but not in Pnmt-KO mice. Isoproterenol and terbutaline induced concentration-dependent increases in tritium overflow in WT mice only. β2-Adrenoceptor protein density was decreased in membrane aorta homogenates of Pnmt-KO mice, and this finding was supported by immunofluorescence confocal microscopy. In conclusion, epinephrine is crucial for β2-adrenoceptor–mediated vasodilation and facilitation of norepinephrine release. In the absence of epinephrine, β2-adrenoceptor protein density was decreased in aorta cell membranes, thus potentially hindering its functional activity.

Blunted renal dopaminergic system in a mouse model of diet-induced obesity.

Obesity has reached epidemic proportions in the Western world and is implicated in the pathophysiology of essential hypertension. The aim of the present study was to evaluate sodium handling, blood pressure and renal dopaminergic system activity in a mouse model of obesity induced by exposure to a hypercaloric diet. From six to 18 weeks of age, animals were fed with a control diet or a high-fat high-simple-carbohydrate (HFHSC) diet. Renal function, blood pressure and urinary and plasmatic catecholamines and biochemical parameters were evaluated in both groups. In parallel, the effects of high sodium intake (HS, 1.0% NaCl, 3 days) on natriuresis, urinary catecholamine excretion and aromatic l-amino acid decarboxylase (AADC) activity were evaluated in control and obese mice. Mice exposed to the HFHSC diet presented obesity, hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia and increased blood pressure. This was accompanied, in obese mice, by decreases in urinary excretion of dopamine and metabolites as well as reduced AADC activity in renal tissues. During HS intake, absolute urinary dopamine excretion increased in control, but not in obese mice. This was accompanied in obese mice by a natriuretic resistance on day 1 of the HS diet. In addition, obese mice presented increased urinary and plasmatic noradrenaline levels, as well as an increased heart rate when compared with control mice. In conclusion, in this model of diet-induced obesity hyperinsulinemia, insulin resistance and increased sympathetic tone are associated with blunted renal dopaminergic activity. It is suggested that this may contribute to compromised sodium excretion and increased blood pressure in obesity.

Renal Dopaminergic System Activity in Uninephrectomized Rats up to 26 Weeks after Surgery

Background: Dopamine of renal origin exerts natriuretic and diuretic effects by activating D1-like receptors located at various regions in the nephron. Two weeks after uninephrectomy the renal dopaminergic system was suggested to be involved in the adaptative increase of sodium excretion. Aim: The aim of the present study was to evaluate the renal adaptations in sodium handling and renal dopaminergic system activity in uninephrectomized (Unx) rats up to 26 weeks after the surgery. Results: A time-dependent increase in both systolic and diastolic blood pressure was observed in Unx rats up to 26 weeks after uninephrectomy. This was accompanied by a compensatory increase in aromatic L-amino acid decarboxylase at 2 weeks but not 10 and 26 weeks after uninephrectomy. In contrast to what has been found 2 weeks after uninephrectomy, at 10 and 26 weeks after surgery the natriuretic response to volume expansion was reduced in Unx rats and this was accompanied by insensitivity of natriuresis to dopamine D1 receptor selective antagonist (Sch23390). Conclusion: A time-dependent decrease in dopamine sensitive natriuresis is observed in Unx rats throughout the 26 weeks after uninephectomy. It is suggested that this may contribute to compromise sodium excretion and increase blood pressure.

Epinephrine increases contextual learning through activation of peripheral β2-adrenoceptors

RationalePhenylethanolamine-N-methyltransferase knockout (Pnmt-KO) mice are unable to synthesize epinephrine and display reduced contextual fear. However, the precise mechanism responsible for impaired contextual fear learning in these mice is unknown.ObjectivesOur aim was to study the mechanism of epinephrine-dependent contextual learning.MethodsWild-type (WT) or Pnmt-KO (129x1/SvJ) mice were submitted to a fear conditioning test either in the absence or in the presence of epinephrine, isoprenaline (non-selective β-adrenoceptor agonist), fenoterol (selective β2-adrenoceptor agonist), epinephrine plus sotalol (non-selective β-adrenoceptor antagonist), and dobutamine (selective β1-adrenoceptor agonist). Catecholamines were separated by reverse-phase HPLC and quantified by electrochemical detection. Blood glucose was measured by coulometry.ResultsRe-exposure to shock context induced higher freezing in WT and Pnmt-KO mice treated with epinephrine and fenoterol than in mice treated with vehicle. In addition, freezing response in Pnmt-KO mice was much lower than in WT mice. Freezing induced by epinephrine was blocked by sotalol in Pnmt-KO mice. Epinephrine and fenoterol treatment restored glycemic response in Pnmt-KO mice. Re-exposure to shock context did not induce a significant difference in freezing in Pnmt-KO mice treated with dobutamine and vehicle.ConclusionsAversive memories are best retained if moderately high plasma epinephrine concentrations occur at the same moment as the aversive stimulus. In addition, epinephrine increases context fear learning by acting on peripheral β2-adrenoceptors, which may induce high levels of blood glucose. Since glucose crosses the blood–brain barrier, it may enhance hippocampal-dependent contextual learning.