Ivone B. Oliveira

Salt-Induced Cardiac Hypertrophy and Interstitial Fibrosis Are Due to a Blood Pressure–Independent Mechanism in Wistar Rats

High salt intake is a known cardiovascular risk factor and is associated with cardiac alterations. To better understand this effect, male Wistar rats were fed a normal (NSD: 1.3% NaCl), high 4 (HSD4: 4%), or high 8 (HSD8: 8%) salt diet from weaning until 18 wk of age. The HSD8 group was subdivided into HSD8, HSD8+HZ (15 mg . kg(-1) . d(-1) hydralazine in the drinking water), and HSD8+LOS (20 mg . kg(-1) . d(-1) losartan in the drinking water) groups. The cardiomyocyte diameter was greater in the HSD4 and HSD8 groups than in the HSD8+LOS and NSD groups. Interstitial fibrosis was greater in the HSD4 and HSD8 groups than in the HSD8+HZ and NSD groups. Hydralazine prevented high blood pressure (BP) and fibrosis, but not cardiomyocyte hypertrophy. Losartan prevented high BP and cardiomyocyte hypertrophy, but not fibrosis. Angiotensin II type 1 receptor (AT(1)) protein expression in both ventricles was greater in the HSD8 group than in the NSD group. Losartan, but not hydralazine, prevented this effect. Compared with the NSD group, the binding of an AT(1) conformation-specific antibody that recognizes the activated form of the receptor was lower in both ventricles in all other groups. Losartan further lowered the binding of the anti-AT(1) antibody in both ventricles compared with all other experimental groups. Angiotensin II was greater in both ventricles in all groups compared with the NSD group. Myocardial structural alterations in response to HSD are independent of the effect on BP. Salt-induced cardiomyocyte hypertrophy and interstitial fibrosis possibly are due to different mechanisms. Evidence from the present study suggests that salt-induced AT(1) receptor internalization is probably due to angiotensin II binding.

Perinatal salt restriction : A new pathway to programming adiposity indices in adult female Wistar rats

Low birth weight has been associated with increased obesity in adulthood. It has been shown that dietary salt restriction during intrauterine life induces low birth weight and insulin resistance in adult Wistar rats. The present study had a two-fold objective: to evaluate the effects that low salt intake during pregnancy and lactation has on the amount and distribution of adipose tissue; and to determine whether the phenotypic changes in fat mass in this model are associated with alterations in the activity of the renin-angiotensin system. Maternal salt restriction was found to reduce birth weight in male and female offspring. In adulthood, the female offspring of dams fed the low-salt diet presented higher adiposity indices than those seen in the offspring of dams fed a normal-salt diet. This was attributed to the fact that adipose tissue mass (retroperitoneal but not gonadal, mesenteric or inguinal) was greater in those rats than in the offspring of dams fed a normal diet. The adult offspring of dams fed the low-salt diet, compared to those dams fed a normal-salt diet, presented the following: plasma leptin levels higher in males and lower in females; plasma renin activity higher in males but not in females; and no differences in body weight, mean arterial blood pressure or serum angiotensin-converting enzyme activity. Therefore, low salt intake during pregnancy might lead to the programming of obesity in adult female offspring.

Usefulness of a quick decalcification of bone sections embedded in methyl metacrylate: an improved method for immunohistochemistry

Immunohistochemistry of undecalcified bone sections embedded in methyl methacrylate (MMA) is not commonly employed because of potential destruction of tissue antigenicity by highly exothermic polymerization. The aim of the present study was to describe a new technique in which a quick decalcification of bone sections embedded in MMA improves the results for immunohistochemistry. The quality of interleukin 1α (IL-1α) immunostaining according to the present method was better than the conventional one. Immunostaining for osteoprotegerin (OPG) and the receptor activator of NF-κB ligand (RANKL) in bone sections of chronic kidney disease patients with mineral bone disorders (CKD-MBD) was stronger than in controls (postmortem healthy subjects). The present study suggested that this method is easy, fast, and effective to perform both histomorphometry and immunohistochemistry in the same bone fragment, yielding new insights into pathophysiological aspects and therapeutic approaches in bone disease.

Salt intake during pregnancy alters offspring’s myocardial structure

BACKGROUND AND AIM To evaluate the effects of low or high salt intake during pregnancy on left ventricle of adult male offspring. METHODS AND RESULTS Low- (LS, 0.15%), normal- (NS, 1.3%) or high-salt (HS, 8% NaCl) diet was given to Wistar rats during pregnancy. During lactation all dams received NS as well as the offspring after weaning. To evaluate cardiac response to salt overload, 50% of each offspring group was fed a high-salt (hs, 4% NaCl) diet from the 21st to the 36th week of age (LShs, NShs, HShs). The remaining 50% was maintained on NS (LSns, NSns and HSns). Echocardiography was done at 20 and 30 weeks of age. Mean blood pressure (MBP), histology and left ventricular angiotensin II content (AII) were analyzed at 36 weeks of age. Interventricular septum, left ventricular posterior wall and relative wall thickness increased from the 20th to the 30th week of age only in HShs, cardiomyocyte mean volume was higher in HShs compared to NShs, LShs and HSns. AII and left ventricular fibrosis were not different among groups. CONCLUSIONS HS during pregnancy programs adult male offspring to a blood pressure and angiotensin II independent concentric left ventricular hypertrophy, with no fibrosis, in response to a chronic high-salt intake.

High-Salt Intake Induces Cardiomyocyte Hypertrophy in Rats in Response to Local Angiotensin II Type 1 Receptor Activation

Many studies have shown that risk factors that are independent of blood pressure (BP) can contribute to the development of cardiac hypertrophy (CH). Among these factors, high-salt (HS) intake was prominent. Although some studies have attempted to elucidate the role of salt in the development of this disease, the mechanisms by which salt acts are not yet fully understood. Thus, the aim of this study was to better understand the mechanisms of CH and interstitial fibrosis (IF) caused by HS intake. Male Wistar rats were divided into 5 groups according to diet [normal salt (NS; 1.27% NaCl) or HS (8% NaCl)] and treatment [losartan (LOS) (HS+LOS group), hydralazine (HZ) (HS+HZ group), or N-acetylcysteine (NAC) (HS+NAC group)], which was given in the drinking water. Tail-cuff BP, transverse diameter of the cardiomyocyte, IF, angiotensin II type 1 receptor (AT1) gene and protein expression, serum aldosterone, cardiac angiotensin II, cardiac thiobarbituric acid-reactive substances, and binding of conformation-specific anti-AT1 and anti-angiotensin II type 2 receptor (AT2) antibodies in the 2 ventricles were measured. Based on the left ventricle transverse diameter data, the primary finding was the occurrence of significant BP-independent CH in the HS+HZ group (96% of the HS group) and a partial or total prevention of such hypertrophy via treatment with NAC or LOS (81% and 67% of the HS group, respectively). The significant total or partial prevention of IF using all 3 treatments (HS+HZ, 27%; HS+LOS, 27%; and HS+NAC, 58% of the HS group, respectively), and an increase in the AT1 gene and protein expression and activity in groups that developed CH, confirmed that CH occurred via the AT1 in this experimental model. Thus, this study unveiled some relevant previously unknown mechanisms of CH induced by chronic HS intake in Wistar rats. The link of oxidative stress with CH in our experimental model is very interesting and stimulates further evaluation for its full comprehension.

Glucose metabolism and hepatic Igf1 DNA methylation are altered in the offspring of dams fed a low-salt diet during pregnancy.

A low-salt (LS) diet during pregnancy has been linked to insulin resistance in adult offspring, at least in the experimental setting. However, it remains unclear if this effect is due to salt restriction during early or late pregnancy. To better understand this phenomenon, 12-week-old female Wistar rats were fed a LS or normal-salt (NS) diet during gestation or a LS diet during either the first (LS10) or second (LS20) half of gestation. Glucose tolerance test, HOMA-IR, gene expression analysis and DNA methylation measurements were conducted for the Insr, Igf1, Igf1r, Ins1 and Ins2 genes in the livers of neonates and in the liver, white adipose tissue and muscle of 20-week-old male offspring. Birth weight was lower in the LS20 and LS animals compared with the NS and LS10 rats. In the liver, the Igf1 levels in the LS10, LS20 and LS neonates were lower than those in the NS neonates. Methylation of the Insr, Igf1r, Ins1 and Ins2 genes was influenced in a variable manner by low salt intake during pregnancy. Increased liver Igf1 methylation was observed in the LS and LS20 neonates compared with their NS and LS10 counterparts. Glucose intolerance was observed in adult offspring as an effect of low salt intake over the duration of pregnancy. Compared to the NS animals, the HOMA-IR was higher in the 12-week-old LS and 20-week-old LS-10 rats. Based on these results, it appears that the reason a LS diet during pregnancy induces a low birth weight is its negative correlation with Igf1 DNA methylation in neonates.

Maternal high-sodium intake alters the responsiveness of the renin-angiotensin system in adult offspring

AIMS The goal of the current study was to evaluate the impact of maternal sodium intake during gestation on the systemic and renal renin-angiotensin-aldosterone-system (RAAS) of the adult offspring. MAIN METHODS Female Wistar rats were fed high- (HSD-8.0% NaCl) or normal-sodium diets (NSD-1.3% NaCl) from 8 weeks of age until the delivery of their first litter. After birth, the offspring received NSD. Tail-cuff blood pressure (TcBP) was measured in the offspring between 6 and 12 weeks of age. At 12 weeks of age, the offspring were subjected to either one week of HSD or low sodium diet (LSD-0.16% NaCl) feeding to evaluate RAAS responsiveness or to acute saline overload to examine sodium excretory function. Plasma (PRA) and renal renin content (RRC), serum aldosterone (ALDO) levels, and renal cortical and medullary renin mRNA expression levels were evaluated at the end of the study. KEY FINDINGS TcBP was higher among dams fed HSD, but no TcBP differences were observed among the offspring. Male offspring, however, exhibited increased TcBP after one week of HSD feeding, and this effect was independent of maternal diet. Increased RAAS responsiveness to the HSD and LSD was also observed in male offspring. The baseline levels of PRA, ALDO, and cortical and medullary renin gene expression were lower but the RRC levels were higher among HSD-fed male offspring (HSDoff). Conversely, female HSDoff showed reduced sodium excretion 4 h after saline overload compared with female NSDoff. SIGNIFICANCE High maternal sodium intake is associated with gender-specific changes in RAAS responsiveness among adult offspring.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

Exposure to fine particulate matter in the air alters placental structure and the renin-angiotensin system

Transforming growth factor beta 1 (TGFβ1), the uteroplacental renin-angiotensin system (RAS) and vascular endothelial growth factor A (VEGF-A) participate in the placentation process. The aim of this study was to investigate the effects of exposure to pollutants on the placenta. Methods Female Wistar rats were exposed to filtered air (F) or to concentrated fine particulate matter (P) for 15 days. After mating, the rats were divided into four groups and again exposed to F or P (FF, FP, PF, PP) beginning on day 6 of pregnancy. At embryonic day 19, the placenta was collected. The placental structure, the protein and gene expression of TGFβ1, VEGF-A, and its receptor Flk-1 and RAS were evaluated by indirect ELISA and quantitative real-time PCR. Results Exposure to P decreased the placental mass, size, and surface area as well as the TGFβ1, VEGF-A and Flk-1 content. In the maternal portion of the placenta, angiotensin II (AngII) and its receptors AT1 (AT1R) and AT2 (AT2R) were decreased in the PF and PP groups. In the fetal portion of the placenta, AngII in the FP, PF and PP groups and AT2R in the PF and PP groups were decreased, but AT1R was increased in the FP group. VEGF-A gene expression was lower in the PP group than in the FF group. Conclusions Exposure to pollutants before and/or during pregnancy alters some characteristics of the placenta, indicating a possible impairment of trophoblast invasion and placental angiogenesis with possible consequences for the maternal-fetal interaction, such as a limitation of fetal nutrition and growth.

High and Low Salt Intake during Pregnancy: Impact on Cardiac and Renal Structure in Newborns

Introduction Previous studies from our laboratory demonstrated that dietary salt overload and salt restriction during pregnancy were associated with cardiac and renal structural and/or functional alterations in adult offspring. The present study evaluated renal and cardiac structure and the local renin-angiotensin system in newborns from dams fed high-, normal- or low-salt diets during pregnancy. Methods Female Wistar rats were fed low- (LS, 0.15% NaCl), normal- (NS, 1.3% NaCl) or high- (HS, 8% NaCl) salt diets during pregnancy. Kidneys and hearts were collected from newborns (n = 6-8/group) during the first 24 hours after birth to evaluate possible changes in structure using stereology. Protein expression of renin-angiotensin system components was evaluated using an indirect enzyme-linked immunosorbent assay (ELISA). Results No differences between groups were observed in total renal volume, volume of renal compartments or number of glomeruli. The transverse diameter of the nuclei of cardiomyocytes was greater in HS than NS males in the left and right ventricles. Protein expression of the AT1 receptor was lower in the kidneys of the LS than in those of the NS and HS males but not females. Protein expression of the AT2 receptor was lower in the kidneys of the LS males and females than in those of the NS males and females. Conclusion High salt intake during pregnancy induced left and right ventricular hypertrophy in male newborns. Salt restriction during pregnancy reduced the expression of renal angiotensin II receptors in newborns.