Angiotensinergic and GABAergic transmission in the medial preoptic area: role in urinary bladder and cardiovascular control in female rats.

Introduction: The medial preoptic area (mPOA) participates in thermoregulatory control and blood pressure modulation as shown by studies with electrical stimulation of this area or cobalt chloride injection, a non-selective synapse inhibitor. This study aimed to investigate whether angiotensin II (Ang II) and GABA could act or not in the mPOA to mediate the cardiovascular and micturition control pathways. Methods: Female Wistar rats were submitted to stereotaxic surgery for implantation of a guide cannula into the mPOA 7 days prior to the experiments. Afterwards, the animals were isoflurane- anesthetized and submitted to the catheterization of the femoral artery and vein and urinary bladder cannulation for mean arterial pressure (MAP), heart rate (HR), and intravesical pressure (IP) recordings, respectively. After the baseline MAP, HR, and IP recordings for 15 min, Ang II (0.1 nM, 1 µL), losartan (AT-1 receptor antagonist, 100 nM, 1 µL), GABA (50 mM, 1 µL) or saline (1 µL) were injected into the mPOA, and the variables were measured for additional 30 min. In a different group of rats, the AT-1 receptor, angiotensin II converting enzyme (ACE), and GABAa receptor gene expression was evaluated in mPOA samples by qPCR. The data are as mean ± SEM and submitted to One-way ANOVA (Tukey posttest) or paired Student t-test (P <0.05). Results: The injection of Ang II into the mPOA evoked a significant hypotension (-37±10 mmHg, n = 6, p = 0.024) and bradycardia (-47 ± 20 bpm, p = 0.030) compared to saline (+1 ± 1 mmHg and +6 ± 2 bpm, n = 6). A significant increase in IP was observed after Ang II injection into the mPOA (+72.25 ± 17.91%, p = 0.015 vs. -1.80 ± 2.98%, n = 6, saline). No significant changes were observed in MAP, HR and IP after the losartan injection in the mPOA compared to saline injection. Injection of GABA into the mPOA evoked a significant fall in MAP and HR (-68 ± 2 mmHg, n = 6, p < 0.0001 and -115 ± 14 bpm, n = 6, p = 0.0002 vs. -1 ± 1 mmHg and +4 ± 2 bpm, n = 6, saline), but no significant changes were observed in IP. The AT-1 receptor, ACE and GABAa receptor mRNA expression was observed in all mPOA samples. Discussion: Therefore, in female rats, Ang II mediated transmission in the mPOA is involved in the cardiovascular regulation and in the control of central micturition pathways. A phasic control dependent on AT-1 receptors in the mPOA seems to be involved in the regulation of those cardiovascular and intravesical 3 parameters. In contrast, GABAergic transmission in the mPOA participates in the pathways of cardiovascular control in anesthetized female rats, nevertheless, this neurotransmission is not involved in the micturition control.

GABAergic and glutamatergic transmission reveals novel cardiovascular and urinary bladder control features in the shell nucleus accumbens.

The shell Nucleus Accumbens (NAcc) projects to the lateral preoptic area, which is involved in the central micturition control and receives inputs from medullary areas involved in cardiovascular control. We investigated the role of GABAergic and glutamatergic transmission in the shell NAcc on intravesical pressure (IP) and cardiovascular control. Male Wistar rats with guide cannulas implanted bilaterally in the shell NAcc 7 days prior to the experiments were anesthetized with 2% isoflurane in 100% O2 and subjected to cannulation of the femoral artery and vein for mean arterial pressure (MAP) and heart rate recordings (HR) and infusion of drugs, respectively. The urinary bladder (UB) was cannulated for IP measurement. A Doppler flow probe was placed around the renal arterial for renal blood flow (RBF) measurement. After the baseline MAP, HR, IP and RBF recordings for 15 min, GABA or bicuculline methiodate (BMI) or L-glutamate or kynurenic acid (KYN) or saline (vehicle) were bilaterally injected into the shell NAcc and the variables were measured for 30 min. Data are as mean ± SEM and submitted to Student́s t test. GABA injections into the shell NAcc evoked a significant fall in MAP and HR and increased IP and RC compared to saline. L-glutamate in the shell NAcc increased MAP, HR and IP and reduced RC. Injections of BMI and KYN elicited no changes in the variables recorded. Therefore, the GABAergic and glutamatergic transmissions in neurons in the shell NAcc are involved in the neural pathways responsible for the central cardiovascular control and UB regulation.

Blockade of vasopressin receptors reduces the threshold pressure of micturition reflex in female rats.

The mechanisms involved in urinary bladder control are not fully understood, but it is well accepted that a complex central network is involved in micturition control. The micturition reflex can be modulated by direct cortical influence through facilitatory and inhibitory mechanisms. In addition, humoral mechanisms are involved in the bladder control. Vasopressin increases bladder contraction and intravesical pressure. This study sought to investigate the effect of intravenous injections of vasopressin receptor antagonists on cystometric parameters in anesthetized female rats. Isoflurane anesthetized adult female Wistar rats underwent femoral artery and vein cannulation for arterial pressure (AP) and heart rate (HR) recordings, and infusion of drugs, respectively. The bladder was also cannulated for intravesical pressure (IP) recordings and infusion of saline (10 mL/h) for cystometric evaluation. After baseline AP, HR and IP recordings, saline (vehicle, 1 mL/kg), V1a (5 µg/kg) or V2 receptor antagonist (5 µg/kg) was injected i.v. and after 25 min the cystometry was carried out. Neither saline nor V1a or V2 receptor blockade evoked any change in AP, HR and IP. Nevertheless, during cystometry, the threshold pressure of the micturition reflex was significantly reduced in rats with V1a (to 19.30 ± 2.39 mmHg) and V2 receptor blockade (to 19.88 ± 2.49 mmHg) compared to the saline group (28.85 ± 2.06 mmHg, p = 0.014). No difference was observed in the other cystometric parameters. Therefore, the data suggest that blockade of V1a and V2 receptors reduces the threshold pressure of the micturition reflex and does not influence other cystometric parameters in anesthetized female Wistar rats.

Activation of 5-HT3 receptors in the medulla oblongata is involved in the phasic control of urinary bladder.

The control of micturition depends on reflex mechanisms, however, it undergoes modulation from cortex, pons and medullary areas. This study investigated if the activation of 5-HT3 receptors in the medulla influences the urinary bladder (UB) regulation in rats. Isoflurane female Wistar rats were submitted to catheterization of the femoral artery and vein for mean arterial pressure (MAP) and heart rate (HR) recordings and injection of drugs, respectively. The UB was cannulated for intravesical pressure (IP) measurement. The Doppler flow probe was placed around the left renal artery for renal conductance (RC) recordings. Phenylbiguanide (PB) and granisetron (GN) were injected into the 4th brain ventricle in rats with guide cannulas implanted 5 days prior to the experiments; or PB and GN were randomly injected intravenously or applied topically (in situ) on the UB. PB injection into 4th V significantly increased IP (68.67 ± 11.70%) and decreased MAP (-29 ± 6 mmHg) compared to saline (0.34 ± 0.64% and -2 ± 2 mmHg), with no changes in the HR and RC. GN injection into the 4th V did not significantly change the IP and RC compared to saline, nevertheless, significantly increased MAP (25 ± 4 mmHg) and heart rate (36 ± 9 bpm) compared to saline. Intravenous PB and GN only produced cardiovascular effects, whilst PB but not GN in situ on the UB evoked increase in IP (111.60 ± 30.36%). Therefore, the activation of 5HT-3 receptors in medullary areas increases the intravesical pressure and these receptors are involved in the phasic control of UB. In contrast, 5-HT3 receptors in the medulla oblongata are involved in the pathways of the tonic control of the cardiovascular system. The activation of 5-HT3 receptors in the bladder cause increase in intravesical pressure and this regulation seem to be under phasic control as the blockade of such receptors elicits no changes in baseline intravesical pressure.

Vitamin D supplementation at different doses affects the vagal component of the baroreceptor reflex and the Bezold-Jarisch reflex in eutrophic rats.

Vitamin D has been used to prevent several diseases. The 1,25 (OH) 2D3, the active form of vitamin D (VitD), participates in calcium metabolism, and has direct action in various tissues as those of the cardiovascular system binding to the VitD receptor. We investigated whether the supplementation with different doses of VitD affect or not the resting mean arterial pressure (MAP) and heart rate (HR), heart rate variability (HRV), baroreceptor and Bezold-Jarisch reflexes in eutrophic rats. Adult male Wistar rats were randomly assigned in 4 groups (Control, VitD 15, 250, and 3,750 IU/day, n = 6/group). After 3 days of supplementation, MAP and HR recordings were performed in freely moving rats. Baseline (resting) MAP, HR, and HRV showed no difference in Control and VitD groups. Nevertheless, the index of the baroreceptor reflex showed that the bradycardic component of the baroreflex evoked by a pressor dose of phenylephrine (3 µg/kg of b.w.) in bolus injection had a significant increase in rats supplemented with VitD 15 IU/day for 3 days compared to Control animals. No difference was observed in the index of the baroreflex evaluated with phenylephrine in rats treated with VitD 250 and 3,750 IU/day for 3 days in comparison to the Control group. The index of the baroreceptor reflex evaluated with an intravenous bolus injection of a depressor dose of sodium nitroprusside (30 µg/kg of b.w.) showed that the tachycardic component of the baroreflex is not different comparing all groups supplemented with VitD and Control animals. Rats supplemented with VitD 15 IU/day presented exaggerated bradycardic responses to the intravenous injection of phenylbiguanide (PBG, 5 µg/kg of b.w.) compared to Control animals, despite the similar hypotension in both groups. Higher doses of supplementation of VitD (250 and 3,750 IU/day for 3 days) abolished the hypotension and bradycardia induced by PBG. The findings suggest that the supplementation with different doses of VitD (15, 250, and 3,750 IU/day) for 3 days did not affect the resting arterial pressure, heart rate and autonomic modulation on the heart in rats. Despite that, the supplementation with a low dose of VitD (15 IU/day for 3 days) improved the sensitivity of the bradycardic component of the baroreflex, whereas higher doses of supplementation with VitD (250 and 3,750 IU/day for 3 days) were unable to cause such effect. In addition, the Bezold-Jarisch reflex responses can be affected regardless the dose of VitD (15, 250 or 3,750 IU/day) supplementation for 3 days in rats.

Unveiling the Angiotensin-(1-7) Actions on the Urinary Bladder in Female Rats.

Angiotensin-(1-7) is a peptide produced by different pathways, and regardless of the route, the angiotensin-converting enzyme 2 (ACE-2) is involved in one of the steps of its synthesis. Angiotensin-(1-7) binds to Mas receptors localized in different cells throughout the body. Whether angiotensin-(1-7) exerts any action in the urinary bladder (UB) is still unknown. We investigated the effects of intravenous and topical (in situ) administration of angiotensin-(1-7) on intravesical pressure (IP) and cardiovascular variables. In addition, the Mas receptors and ACE-2 gene and protein expression were analyzed in the UB. Adult female Wistar rats were anesthetized with 2% isoflurane in 100% O2 and submitted to the catheterization of the femoral artery and vein for mean arterial pressure (MAP) and heart rate (HR) recordings, and infusion of drugs, respectively. The renal blood flow was acquired using a Doppler flow probe placed around the left renal artery and the renal conductance (RC) was calculated as a ratio of Doppler shift (kHz) and MAP. The cannulation of the UB was performed for IP recording. We observed that angiotensin-(1-7) either administered intravenously [115.8 ± 28.6% angiotensin-(1-7) vs. -2.9 ± 1.3% saline] or topically [147.4 ± 18.9% angiotensin-(1-7) vs. 3.2 ± 2.8% saline] onto the UB evoked a significant (p < 0.05) increase in IP compared to saline and yielded no changes in MAP, HR, and RC. The marked response of angiotensin-(1-7) on the UB was also investigated using quantitative real-time polymerase chain reaction and western blotting assay, which demonstrated the mRNA and protein expression of Mas receptors in the bladder, respectively. ACE-2 mRNA and protein expression was also observed in the bladder. Therefore, the findings demonstrate that angiotensin-(1-7) acts in the UB to increase the IP and suggest that this peptide can be also locally synthesized in the UB.

Lateral Preoptic Area Neurons Activated by Angiotensin-(1-7) Increase Intravesical Pressure: A Novel Feature in Central Micturition Control.

Central micturition control and urine storage involve a multisynaptic neuronal circuit for the efferent control of the urinary bladder. Electrical stimulation of the lateral preoptic area (LPA) at the level of the decussation of the anterior commissure in cats evokes relaxation of the bladder, whereas ventral stimulation of LPA evokes vigorous contraction. Endogenous Angiotensin-(1-7) [(Ang-(1-7)] synthesis depends on ACE-2, and its actions on binding to Mas receptors, which were found in LPA neurons. We aimed to investigate the Ang-(1-7) actions into the LPA on intravesical pressure (IP) and cardiovascular parameters. The gene and protein expressions of Mas receptors and ACE-2 were also evaluated in the LPA. Angiotensin-(1-7) (5 nmol/µL) or A-779 (Mas receptor antagonist, 50 nmol/µL) was injected into the LPA in anesthetized female Wistar rats; and the IP, mean arterial pressure (MAP), heart rate (HR), and renal conductance (RC) were recorded for 30 min. Unilateral injection of Ang-(1-7) into the LPA increased IP (187.46 ± 37.23%) with peak response at ∼23-25-min post-injection and yielded no changes in MAP, HR, and RC. Unilateral or bilateral injections of A-779 into the LPA decreased IP (-15.88 ± 2.76 and -27.30 ± 3.40%, respectively) and elicited no changes in MAP, HR, and RC. The genes and the protein expression of Mas receptors and ACE-2 were found in the LPA. Therefore, the LPA is an important part of the circuit involved in the urinary bladder control, in which the Ang-(1-7) synthetized into the LPA activates Mas receptors for increasing the IP independent on changes in RC and cardiovascular parameters.

Unravelling the intravenous and in situ vasopressin effects on the urinary bladder in anesthetized female rats: More than one vasopressin receptor subtype involved?

Urinary bladder dysfunctions show high prevalence in women. We focused to investigate the intravenous and in situ (topic) vasopressin effects on the bladder and also to characterize the vasopressin receptor subtypes in the bladder. Adult female Wistar rats anesthetized with isoflurane underwent to the cannulation of the femoral artery and vein, and also urinary bladder for mean arterial pressure, heart rate and intravesical pressure (IP) recordings, respectively. Doppler flow probe was placed around the renal artery for blood flow measurement. After baseline recordings, intravenous injection of saline or vasopressin at different doses (0.25, 0.5, 1.0 ng/ml/kg of b.w.); or 0.1 ml of saline or 0.1 ml of vasopressin at different doses (0.25, 0.5, 1.0 ng/ml) was randomly dropped on the bladder. In another group of rats, the UB was harvest for gene expression by qPCR and also for protein expression by Western blotting of the vasopressin receptor subtypes. We observed that either intravenous or in situ vasopressin evoked a huge increase in the IP in a dose-dependent manner compared to saline, whilst no differences were observed in the cardiovascular parameters. The genes and the protein expression of V1a, V1b and V2 vasopressin receptors subtypes were found in the bladder. Intravenous injection of V1a or V2 receptor antagonist evoked a huge fall in IP and 30 min later, i.v or in situ vasopressin evoked responses on IP were significantly attenuated. Therefore, intravenous or in situ vasopressin increases the IP due to binding in V1a or V2 receptors localized in the bladder.

Body adiposity index assess body fat with high accuracy in nondialyzed chronic kidney disease patients.

OBJECTIVE: High body fat (BF) is an alarming condition that also affects nondialyzed chronic kidney disease (CKD) patients. Distinct methods are used to evaluate BF; however, in CKD population it remains unclear which one is more reliable showing high accuracy. Dual-energy X-ray absorptiometry (DXA), used as reference method to estimate adiposity, is expensive and time consuming to be applied in clinical settings. Recently, a new body adiposity index (BAI), that estimates BF from easily accessible measures, was validated in the general population. The aim of this study was to evaluate which simple and practical method, routinely used to estimate BF, shows the highest accuracy compared with DXA, in nondialyzed CKD patients. DESIGN AND METHODS: In this cross-sectional study BF was estimated by DXA, bioelectrical impedance analysis (BIA), anthropometry (ANTHRO), and BAI. Serum leptin levels were determined. RESULTS: Studied patients (n = 134) were 55% males, 54% overweight/obese, and 64.9 ± 12.5 years old, with estimated glomerular filtration rate (eGFR) = 29.0 ± 12.7 ml/min. The correlation coefficient was higher between DXA vs. ANTHRO (r = 0.76) and BAI (r = 0.61) than with BIA (r = 0.57), after adjusting for gender, age, and eGFR (P < 0.0001). Therefore, the Lin's concordance correlation coefficient and Bland-Altman plots were performed to measure the accuracy (C_b) between DXA with both ANTHRO and BAI. A higher accuracy (C_b = 0.82) and lower mean difference (-3.4%) was observed for BAI than for ANTHRO (C_b = 0.61; -8.4%). Leptin levels correlated (P < 0.0001) with DXA (r = 0.56) and BAI (r = 0.59). CONCLUSIONS: These findings suggest that BAI estimates BF with high accuracy in nondialyzed CKD patients and may be helpful in the treatment of this population with increased BF.

Annual variation in body fat is associated with systemic inflammation in chronic kidney disease patients Stages 3 and 4: a longitudinal study.

BACKGROUND: In dialysis patients, cross-sectional studies show that total and abdominal body fat associate with inflammatory markers. Whether this is true in earlier disease stages is unknown. We evaluated the cross-sectional and longitudinal (12-month interval) association between body fat markers and C-reactive protein (CRP) in pre-dialysis chronic kidney disease (CKD) patients. METHODS: We studied, over a period of 1 year, clinically stable CKD patients at Stages 3-4 who were under treatment in a single outpatient clinic. Fifty-seven patients were included and 44 concluded the observational period [males: 66%; age: 62.9±13.9 years; body mass index (BMI): 25.5±5.1 kg/m2; estimated glomerular filtration rate (eGFR): 34±12.3 mL/min/1.73 m2]. Total body fat (skinfold thicknesses), waist circumference (WC), laboratory measurements (serum creatinine, total cholesterol, albumin, high-sensitivity CRP and leptin) and food intake (24-h food recall) were assessed at baseline and after 12±2 months. RESULTS: Most patients had anthropometric parameters in the range of overweight/obesity and none had signs of protein-energy wasting. In univariate analysis, changes (delta: end-baseline) in CRP were associated (P<0.05) with changes in BMI (r=0.39) and WC (r=0.33). In multiple regression analysis, these associations remained significant (P<0.05) even after adjusted by potential confounders (sex, diabetes, baseline age and eGFR). CONCLUSIONS: During a follow-up of 12 months, changes in BMI and WC were directly associated with changes in CRP. Our results support the concept that interventions aimed at reducing weight and/or abdominal adiposity in pre-dialysis CKD patients may also translate into reduced systemic inflammation.

Agreement between anthropometry and bioelectrical impedance for measuring body fat in nonobese and obese nondialyzed chronic kidney disease patients.

OBJECTIVES: We investigated reproducibility and agreement between anthropometry (ANT) and bioelectrical impedance (BIA) in nonobese and obese nondialyzed chronic kidney disease (CKD) patients, and examined the factors influencing the agreement between these two methods. PATIENTS AND METHODS: One hundred and five clinically stable CKD patients, comprising stages 3 and 4 of CKD, had their body fat (BF) assessed by ANT and BIA. Patients were stratified into nonobese (body mass index [BMI] <25 kg/m(2), n = 48, 26 males, aged 64.5 +/- 13.6 years) and overweight/obese (BMI > or =25 kg/m(2), n = 57, 35 males, aged 63.1 +/- 12.1 years; +/-SD in all values) groups. RESULTS: In the nonobese group, BF as assessed by ANT was similar to body fat as assessed by BIA (11.4 +/- 3.8 kg vs 11.7 +/- 4.1 kg, respectively, no significance). The concordance correlation coefficient (CCC) was indicative of good reproducibility for males (0.67; 95% confidence interval [CI], 0.41 to 0.83) and females (0.88; 95% CI, 0.71 to 0.95), and the mean inter-method difference between ANT and BIA and limits of agreement were -0.9 (-6.8 to 4.9) kg for males, and 0.4 (-3.4 to 4.3) kg for females. In the overweight/obese group, a weak CCC was observed for males (0.46; 95% CI, 0.26 to 0.62) and females (0.53; 95% CI, 0.29 to 0.70), and the mean inter-method difference between both methods was 5.7 (-3.2 to 14.7) kg for males, and 6.4 (-1.8 to 14.7) kg for females. In the multiple regression analysis (adjusted r(2) = 0.42, n = 105), BMI, sex, body water, and age were independent determinants of the agreement between ANT and BIA. CONCLUSIONS: For nonobese patients, ANT and BIA can be applied equally well for assessing BF. For overweight/obese patients a weak agreement was observed. Body mass index, sex, body water, and age were the factors influencing the agreement between ANT and BIA.

Tratamento da doença renal crônica: estratégias para o maior envolvimento do paciente em seu auto-cuidado / Chronic kidney disease treatment: strategies for increasing the patient’s participation in self-care

A identificação de pacientes portadores de doença renal crônica (DRC) ou em risco para o seu desenvolvimento pode ser feita por meio de testes simplesna rotina clínica. A importância do diagnóstico precoce da DRC está na possibilidade de instituição de intervenções clínicas que contribuem para o retardo da progressão da doença, postergando o início da terapia renal substitutiva e, portanto, trazem ganhos para a qualidade de vida do paciente, contribuindo com redução de custos do sistema público de saúde. No entanto, uma parcela importante dos pacientes é encaminhada tardiamente ao nefrologista. Mesmo com o encaminhamento tardio, o tratamento da doença permite o controle da condição clínica do paciente. As diversas complicações que ocorremao longo da evolução da DRC chamam atenção para a importância do tratamento especializado e multifacetado, realizado por equipe interdisciplinar. A baixa adesão dos pacientes ao tratamento de doenças crônicas reforça a importância desse tipo de abordagem. Neste sentido, o presente trabalho tem como objetivos apresentar estratégias que possam envolver mais os pacientes no seu auto-cuidado, aumentar a adesão ao tratamento e otimizar o trabalhoda equipe interdisciplinar.

Simple and cost-effective laboratorial parameters can be used by the clinical practitioner for screening and diagnosing chronic kidney disease (CKD). The importance of diagnosing CKD, still in early stages, lies in the possibility of delaying progression of the disease, postponing the beginning of renal replacement therapy, and therefore, improving the patient’s quality of life and lowering costs of the public health care system. In addition, early detection of CKD allows for better control of the disturbances commonly developed as renal function decreases. However, an important proportion of patients are referred to the nephrologists only when the kidney function is markedly decreased. Even considering this late referral, treatment before the beginning of renal replacement therapy allows for better control of the patient’s overall clinical state. Such treatment can achieve better results when a multidisciplinary team is present. This is particularly important when considering multiple aspects of CKD and the low adherence of the patients to the treatment of chronic diseases in general. Accordingly, this study proposes a series of strategies to enhance the patient’s adherence to the treatment and, ultimately, improve the results and patient outcome.