Elton Dias da Silva

Long term treatment with ACE inhibitor enalapril decreases body weight gain and increases life span in rats.

Renin-angiotensin system is involved in homeostasis processes linked to renal and cardiovascular system and recently has been linked to metabolic syndrome. We analyzed the influence of long term angiotensin I converting enzyme (ACE) inhibitor enalapril treatment in normotensive adult Wistar rats fed with standard or palatable hyperlipidic diets. Our results show that long term enalapril treatment decreases absolute food intake, serum leptin concentration and body weight gain. Moreover, in adipose tissue, enalapril treatment led to decreased ACE activity, enhanced the expression of peroxisome proliferator activated receptor gamma, adiponectin, hormone-sensitive lipase, fatty acid synthase, catalase and superoxide dismutase resulting in prolonged life span. On the other hand, the ACE inhibitor was not able to improve the transport of leptin through the blood brain barrier or to alter the sensitivity of this hormone in the central nervous system. The effect of enalapril in decreasing body weight gain was also observed in older rats. In summary, these results extend our previous findings and corroborate data from the literature regarding the beneficial metabolic effects of enalapril and show for the first time that this ACE inhibitor prolongs life span in rats also fed with palatable hyperlipidic diet, an action probably correlated with adipose tissue metabolic modulation and body weight reduction.

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B1 receptor knockout mice (B1 −/−) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings Here we show that kinin B1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B1 receptors. In these cells, treatment with the B1 receptor agonist des-Arg9-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B1 −/− mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B1 receptor was limited to cells of the adipose tissue (aP2-B1/B1 −/−). Similarly to B1 −/− mice, aP2-B1/B1 −/− mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B1 −/− mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B1/B1 −/− when compared to B1 −/− mice. When subjected to high fat diet, aP2-B1/B1 −/− mice gained more weight than B1 −/− littermates, becoming as obese as the wild types. Conclusions/Significance Thus, kinin B1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

Effect of kinin B2 receptor ablation on skeletal muscle development and myostatin gene expression

Bradykinin (BK) is an active peptide that binds to the kinin B(2) receptor and induces biological events during the development and adult life. In this study we aimed to investigate the effect of kinin B(2) receptor ablation in the postnatal skeletal muscle development and body composition in adult life. For studies of skeletal muscle development, control (C57Bl6 - WT) and B(2) receptor knockout mice (B(2)(-/-)) were sacrificed at 15, 30 and 90days after birth, the gastrocnemius skeletal muscle was weighed and myostatin gene expression evaluated by real time PCR. For energy balance determination, data from control and B(2)(-/-) at 90 and 120days were collected by calorimetric method. Body composition at 120days was determined by chloroform-methanol (total body fat) and Lowry-modified method (total body protein). The results show that B(2)(-/-) have significantly increased total body weight at 15, 30 and 90days of life, when compared to WT. The weight of the gastrocnemius skeletal muscle was also significantly increased at 30 and 90days of life. Body composition analyses revealed that B(2)(-/-) mice exhibit more total corporal protein and less total corporal fat. Energy balance revealed that B(2)(-/-) have increased metabolizable energy intake and energy expenditure when compared to control mice, resulting in a lower energy gain. Interestingly, myostatin mRNA expression was significantly decreased in 15 and 30days old B(2)(-/-) mice and after icatibant treatment of WT adult mice for 5days. In conclusion, together our results show that kinin B(2) receptor deletion increases lean mass, reduces fat mass and improves metabolism efficiency in mice. The mechanism involved in this phenotype could be related to the reduction of myostatin gene expression during postnatal life.

Targeted Next-Generation Sequencing in Brazilian Children With Nephrotic Syndrome Submitted to Renal Transplant

Background The aims of this study were to identify the genetic mutations profile in Brazilian children with nephrotic syndrome (NS) and to determine a genotype-phenotype correlation in this disease. Methods Next-generation sequencing and mutation analysis were performed on 24 genes related to NS in a cross-sectional study involving 95 children who underwent kidney transplantation due to NS, excluding congenital cases. Results A total of 149 variants were identified in 22 of 24 sequenced genes. The mutations were classified as pathogenic, likely pathogenic, likely benign and benign per the chance of causing the disease. NPHS2 was the most common mutated gene. We identified 8 (8.4%) patients with hereditary NS and 5 (5%) patients with probably genetically caused NS. COL4A3-5 variants were found as well, but it is not clear whether they should be considered isolated FSGS or simply a misdiagnosed type of the Alport spectrum. Considering the clinical results, hereditary NS patients presented a tendency to early disease onset when compared with the other groups (P = 0.06) and time to end stage renal disease (ESRD) was longer in this group (P = 0.03). No patients from hereditary NS group had NS recurrence after transplantation. Conclusions This is the first study in children with steroid-resistant NS who underwent kidney transplantation using next-generation sequencing. Considering our results, we believe this study has shed some light to the uncertainties of genotype-phenotype correlation in NS, where several genes cooperate to produce or even to modify the course of the disease.

Simple Method to Genotype the ACTN3 r577x Polymorphism

The alpha-actinin-3 r577x polymorphism (rs1815739) is one of the most important polymorphisms associated with athletic performance. This single-nucleotide mutation leads to a premature stop codon, resulting in a nonfunctional protein product. The presence of the dominant R allele is associated with full power skeletal muscle contraction. Homozygosity for the X allele is correlated with more efficient energy disposure. Restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) are the standard methods used to genotype this polymorphism, but they are expensive and require special equipments. Here, we present a simple and cost-efficient method to genotype the ACTN3 r577x polymorphism by a single PCR. External primers yield a 690-bp product that indicates the template quality. Internal primers produce a 413-bp product if the R allele is present and a 318-bp product if the X allele is present. Our four-primer genotyping PCR was validated by the standard real-time PCR, generally used to genotype this single-nucleotide polymorphism, demonstrating the accuracy of this method. This protocol is perfect for small- or large-scale cohort genotyping of the ACTN3 r577x polymorphism.

Kinin B1 and B2 receptor deficiency protects against obesity induced by a high-fat diet and improves glucose tolerance in mice.

The kallikrein-kinin system is well known for its role in pain and inflammation, and has been shown recently by our group to have a role also in the regulation of energy expenditure. We have demonstrated that B1 receptor knockout (B1KO) mice are resistant to obesity induced by a high-fat diet (HFD) and that B1 receptor expression in adipocytes regulates glucose tolerance and predisposition to obesity. However, it is also known that in the absence of B1 receptor, the B2 receptor is overexpressed and can take over the function of its B1 counterpart, rendering uncertain the role of each kinin receptor in these metabolic effects. Therefore, we investigated the impact of ablation of each kinin receptor on energy metabolism using double kinin receptor knockout (B1B2KO) mice. Our data show that B1B2KO mice were resistant to HFD-induced obesity, with lower food intake and feed efficiency when compared with wild-type mice. They also had lower blood insulin and leptin levels and higher glucose tolerance after treatment with an HFD. Gene expression for tumor necrosis factor-alpha and C-reactive protein, which are important genes for insulin resistance, was reduced in white adipose tissue, skeletal muscle, and the liver in B1B2KO mice after the HFD. In summary, our data show that disruption of kinin B1 and B2 receptors has a profound impact on metabolic homeostasis in mice, by improving glucose tolerance and preventing HFD-induced obesity. These novel findings could pave the way for development of new pharmacological strategies to treat metabolic disorders such as insulin resistance and obesity.

Novel Complex ABCA4 Alleles in Brazilian Patients With Stargardt Disease: Genotype-Phenotype Correlation

Purpose To analyze the presence of complex alleles of the ABCA4 gene in Brazilian patients with Stargardt disease and to assess the correlation with clinical features. Methods This was an observational cross-sectional study. Patients with a diagnosis of Stargardt disease who presented three pathogenic variants of the ABCA4 gene or who had variants previously described as complex alleles were included. The relatives of these probands were evaluated in the segregation analysis. The patients were evaluated based on age at symptom onset and visual acuity, and the clinical characteristics were classified according to the findings observed on autofluorescence examination. Results Among the 47 families analyzed, approximately 30% (14/47) presented complex alleles. The segregation analysis in 14 families with cases of Stargardt disease identified three novel complex alleles and one previously described complex allele. The known complex allele p.[Leu541Pro; Ala1038Val] was identified in two families. The novel complex alleles identified were p.[Leu541Pro; Arg1443His] in five families, p.[Ser1642Arg; Val1682_Val1686del] in seven families, and p.[Pro1761Arg; Arg2106Cys] in one family. Furthermore, four new variants (p.Lys22Asn, p.Asp915Asn, p.Glu1447Val, and p.Pro1761Arg) were identified in the second allele of the ABCA4 gene. Conclusions Segregation analysis is important in order to confirm the molecular diagnosis of patients with Stargardt disease, given the frequency of complex alleles in the ABCA4 gene. The various pathogenic variation combinations observed in this study were associated with different phenotypes.

Genetic analysis of hereditary angioedema in a Brazilian family by targeted next generation sequencing.

Abstract Hereditary angioedema (HAE) is accompanied by an overproduction of bradykinin (BK) as the primary mediator of swelling. Although many proteins may be involved in regulating the wide spectrum of HAE symptoms, most studies have only focused on C1-INH and FXII. For the first time, a next generation sequencing (NGS) method was applied to develop a robust, time- and cost-effective diagnostic and research tool to analyze selected genes related to HAE. The entire coding region and the exon-intron boundaries of 15 genes from 23 subjects of a Brazilian family, nine of whom were symptomatic, were analyzed by NGS. One new mutation found uniquely in the nine symptomatic patients, p.Ala457Pro in the SERPING1 gene, was estimated as likely to be pathogenic (PolyPhen-2 software analysis) and is the main candidate to be responsible for HAE in these patients. Alterations identified in a few asymptomatic individuals but also found in almost all symptomatic patients, such as p.Ile197Met (HMWK), p.Glu298Asp (NOS3) and p.Gly354Glu (B2R), may also be involved in modulating patient-specific symptoms. This NGS gene panel has proven to be a valuable tool for a quick and accurate molecular diagnosis of HAE and efficient to indicate modulators of HAE symptoms.

PROM1 gene variations in Brazilian patients with macular dystrophy.

ABSTRACT Background: Although the pathogenicity of the prominin-1 (PROM1) gene has already been described as associated with autosomal dominant Stargardt disease, little is known about sequence variations in this gene. Purpose: The aim of this study was to evaluate PROM1 gene sequence variations in patients with macular dystrophy. Material and methods: This retrospective study evaluated variations in the PROM1 gene detected by next-generation sequencing test in patients with macular dystrophy and Stargardt disease. Results: Of 25 medical records of patients with Stargardt disease, three records of patients with PROM1 gene sequence variations were selected for the study. The p.Asp776Val and p.Asp829Asn variants were detected in cases 1 and 2, respectively, and predicted to be pathogenic; they were probably responsible for macular dystrophy in these patients. Case 3 showed a p.Ala643Gly variant in the PROM1 gene and a single variation in the ABCA4 gene, but molecular testing results were inconclusive. Conclusions: In cases of Stargardt disease, where molecular testing results are inconclusive for pathogenic variations in the ABCA4 gene, variations in the PROM1 gene may occur and be considered responsible for the disease in the molecular analysis. This study described three cases in which variations in PROM1 gene may play a role in the pathogenesis of macular dystrophy or be associated with both autosomal recessive and autosomal dominant inheritance.

Early pharmacological inhibition of angiotensin-I converting enzyme activity induces obesity in adulthood

We have investigated early programming of body mass in order to understand the multifactorial etiology of obesity. Considering that the renin-angiotensin system (RAS) is expressed and functional in the white adipose tissue (WAT) and modulates its development, we reasoned whether early transitory inhibition of angiotensin-I converting enzyme activity after birth could modify late body mass development. Therefore, newborn Wistar rats were treated with enalapril (10 mg/kg of body mass) or saline, starting at the first day of life until the age of 16 days. Between days ninetieth and hundred and eightieth, a group of these animals received high fat diet (HFD). Molecular, biochemical, histological, and physiological data were collected. Enalapril treated animals presented hyperphagia, overweight, and increased serum level of triglycerides, total cholesterol and leptin, in adult life. Body composition analyses revealed higher fat mass with increased adipocyte size in these animals. Molecular analyses revealed that enalapril treatment increases neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) gene expression in hypothalamus, fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) gene expression in retroperitoneal WAT, and decreases peroxixome proliferators-activated receptor (PPAR)γ, PPARα, uncoupling protein (UCP)2, and UCP3 gene expression in WAT. The results of the current study indicate that enalapril administration during early postnatal development increases body mass, adiposity and serum lipids in adulthood associated with enhanced food intake and decreased metabolic activity in WAT, predisposing to obesity in adulthood.