Gestational Low Protein Diet Modulation on miRNA Transcriptome and Its Target During Fetal and Breastfeeding Nephrogenesis

BackgroundThe kidney ontogenesis is the most structurally affected by gestational protein restriction, reducing 28% of their functional units. The reduced nephron number is predictive of hypertension and cardiovascular dysfunctions that are generally observed in the adult age of most fetal programming models. We demonstrate miRNAs and predict molecular pathway changes associated with reduced reciprocal interaction between metanephros cap (CM) and ureter bud (UB) and a 28% decreased nephron stem cells in the 17 gestational days (17GD) low protein (LP) intake male fetal kidney. Here, we evaluated the same miRNAs and predicted targets in the kidneys of 21GD and at 7 days of life (7DL) LP offspring to elucidate the molecular modulations during nephrogenesis.MethodsPregnant Wistar rats were allocated into two groups: NP (regular protein diet- 17%) or LP (diet-6%). miRNA transcriptome sequencing (miRNA-Seq) was performed on the MiSeq platform from 21GD and 7DL male offspring kidneys using previously described methods. Among the top 10 dysfunctional regulated miRNAs, we validated 7 related to proliferation, differentiation, and apoptosis processes and investigated predicted target genes and proteins by RT-qPCR and immunohistochemistry.ResultsIn 21GD, LP fetuses were identified alongside 21 differently expressed miRNAs, of which 12 were upregulated and 9 downregulated compared to age-matched NP offspring. In 7-DL LP offspring, the differentially expressed miRNAs were counted to be 74, of which 46 were upregulated and 28 downregulated. The curve from 17-GD to 7-DL shows that mTOR was fundamental in reducing the number of nephrons in fetal kidneys where the mothers were subjected to a protein restriction. IGF1 and TGFβ curves also seemed to present the same mTOR pattern and were modulated by miRNAs 181a-5p, 181a-3p, and 199a-5p. The miRNA 181c-3p modulated SIX2 and Notch1 reduction in 7-DL but not in terms of the enhanced expression of both in the 21-GD, suggesting the participation of an additional regulator. We found enhanced Bax in 21-GD; it was regulated by miRNA 298-5p, and Bcl2 and Caspase-3 were controlled by miRNA (by 7a-5p and not by the predicted 181a-5p). The miRNA 144-3p regulated BCL6, which was enhanced, as well as Zeb 1 and 2 induced by BCL6. These results revealed that in 21GD, the compensatory mechanisms in LP kidneys led to the activation of UB ramification. Besides, an increase of 32% in the CM stem cells and a possible cell cycle halt of renal progenitor cells, which remaining undifferentiated, were observed. In the 7DL, much more altered miRNA expression was found in LP kidneys, and this was probably due to an increased maternal diet content. Additionally, we verified the activation of pathways related to differentiation and consumption of progenitor cells

Functional properties of the protein transduction domais associated ciliary neurotrophic factor : analysis of its effects on energy metabolism regulating hypothalamic regions

Orientadores: Francesco Langone, Licio Augusto VellosoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Fator Neurotrófico Ciliar (CNTF) é uma neurocitocina com múltiplas atividades biológicas, sendo notável sua habilidade de proteger motoneurônios. Entretanto, a administração de CNTF leva à redução de peso corporal. Por outro lado, a administração do CNTF fusionado a um Domínio de Transdução de Proteínas (PTD), denominado TAT-CNTF, é capaz de proteger motoneurônios da medula espinhal axotomizados sem causar este efeito. O presente trabalho investigou se a administração intracerebroventricular (i.c.v.) de TAT-CNTF produz os conhecidos efeitos catabólicos do CNTF. Para isso, ratos Wistar machos, com uma cânula crônicamente implantada no ventrículo lateral, foram distribuídos em quatro grupos: TAT-CNTF (2,5µg/8µl), CNTF (2,5µg/8µl), Leptina (LEP) (5µg/8µl) e PBS, que receberam uma infusão i.c.v. a cada 12h por 4 dias. O grupo tratado com TAT-CNTF apresentou menor perda de peso quando comparado aos grupos CNTF e LEP. As infusões i.c.v. de TAT-CNTF não reduziram o peso das gorduras retroperitonial (RP), epididimal (EP) e marrom interescapular (GM). O grupo CNTF apresentou redução do peso destas gorduras. Os grupos CNTF e LEP apresentaram aumento da fragmentação do DNA nas gorduras RP e EP. Por outro lado, o grupo TAT-CNTF não apresentou aumento da fragmentação do DNA nas amostras de RP, EP e GM. Um padrão de degradação internucleossomal do DNA e a presença de células TUNEL positivas foram detectados apenas nas gorduras dos animais dos grupos CNTF e LEP. Estes grupos também apresentaram aumento da expressão da UCP1 na GM, ao passo que o grupo TAT-CNTF não apresentou tal resultado. A análise da fosforilação da STAT3 no hipotálamo após uma única infusão i.c.v. de TAT-CNTF demonstrou, após 20 minutos, um efeito menor que o observado após infusão de CNTF. Em conclusão, nossos dados sugerem que o TAT-CNTF possui ação diferente do CNTF nas áreas hipotalâmicas envolvidas no controle da ingesta e do metabolismoAbstract: The Ciliary Neurotrophic Factor (CNTF) is a neurocitokine with multiple biological activities, being notable its ability to protect lesioned motoneurons. However, administration of CNTF leads to reduction of body mass, while administration of CNTF fusioned with a Protein Transduction Domain (PTD), named TAT-CNTF, protects lesioned spinal motoneurons with no effects on body weight. In the present work, we investigated whether intracerebroventricular (i.c.v.) administration of TAT-CNTF would produce CNTF known catabolic effects. Male Wistar rats with a canula chronically implanted in the lateral ventricle were randomly assigned to four treatment groups: TAT-CNTF (2,5µg/8µl), CNTF (2,5µg/8µl), Leptin (LEP) (5µg/8µl) and PBS, that received an i.c.v. infusion every 12h for 4 days. TAT-CNTF treated group had a reduced weight loss when compared with CNTF and LEP groups. TAT-CNTF i.c.v. infusions did not reduce the weights of retroperitoneal (RP) and epididimal (EP) white adipose tissue, as well as interescapular brown adipose tissue (BAT) while CNTF i.c.v. administration reduced the weight of all these tissues. CNTF and LEP groups showed an increase of DNA fragmentation in RP and EP. On the other hand, TAT-CNTF group had no increase in DNA fragmentation in RP, EP and GM. A DNA ladder pattern and TUNEL positive cells could only be detected in adipose tissues from CNTF and LEP groups. CNTF and LEP treated groups had an increase in the expression of UCP1 in BAT, while TAT-CNTF treatment had no effects on UCP1 expression. An acute i.c.v. administration demonstrated that after 20 minutes TAT-CNTF induced less intense STAT3 phosphorilation in the hypothalamus when compared with CNTF acute infusions. In conclusion these data suggest that TAT-CNTF has a different action on hypothalamic areas involved in the control of food intake and energy metabolismMestradoFisiologiaMestre em Biologia Funcional e Molecula

Janus Kinase 2 Activation Participates In Prostaglandin E-2-induced Hyperalgesia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Prostaglandin E2 (PGE2) is one of the major signaling molecules involved in hyperalgesia, acting directly on nociceptors and resulting in the activation of PKA and PKC. Once active, these kinases phosphorylate many cellular proteins, resulting in changes on nociceptors sensorial transduction properties. The Janus Kinases (JAKs) are a family of intracellular signaling molecules generally associated with cytokine signaling, and their activity can be increased in nociceptors after peripheral inflammation. However, there are no evidences of JAKs direct involvement in PGE2 mediated sensitization of nociceptors. Therefore, the aim of the present study was to explore a possible role for JAKs in PGE2 mediated sensitization. In cultured dorsal root ganglion (DRG) neurons, we observed that the administration of PGE2 increases capsaicin induced calcium transients, and a pre-incubation of DRG cells with the JAK inhibitor AG490 blocks this PGE2 in vitro effect. Intrathecal administration of AG490 to ten-weeks old male Wistar rats reduces the hyperalgesia induced by the intraplantar administration of PGE2 or carrageenan in the right hind paw. We also observed that carrageenan administration in the right hind paw induced an increase in membrane associated PKCepsilon in the ipsilateral L5 DRG, and this increase was blocked by intrathecal AG490 administration. In conclusion the present study indicates that the JAKs expressed in the DRG and spinal cord may have a role in the sensitization of nociceptors by a peripheral inflammatory event. Moreover, the inhibition of JAKs may be a possible novel pharmacological target for the control of the inflammatory hyperalgesia. (C) 2016 Published by Elsevier Inc.166812Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - CAPESConselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq/Brazil [140621/2008-3]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

The improvement of the anti-hyperalgesic effect of ketamine and of its isomers by the administration of ifenprodil

Intrathecal or epidural administration of NMDA (N-methyl-D-aspartate) receptors antagonists, in special ketamine and ifenprodil are used to control moderate to severe hyperalgesia in humans. Activation of NMDA receptor usually requires binding of two agonists, glutamate and glycine, in different receptor subunits. Ketamine is a NMDA receptor antagonist and acts at phencyclidine site in NR1 subunit while ifenprodil is a selective NR2B subunit antagonist of NMDA receptor. The aim of this study was to investigate the pharmacological interactions between ketamine or its isomers and ifenprodil, when intrathecally co-administrated, to reduce prostaglandin E(2)-induced hyperalgesia in rat's hind paw. The intrathecal administration of ketamine, its isomers R(-) or S(+), or ifenprodil-induced anti-hyperalgesic effects in a dose-related manner. Ifenprodil, in a dose that did not induce significant effect when administrated alone, significantly improved the anti-hyperalgesic effect of ketamine or its isomers. The other way round, ketamine or S(+) ketamine, but not R(-) ketamine, in a dose that did not induce significant effect when administrated alone, improved the anti-hyperalgesic effect of ifenprodil. However, by comparing ED(50)s (half maximal effective doses), ifenprodil-induced potentiation of ketamine was significantly greater than ketamine-induced potentiation of ifenprodil. The findings of this present study suggest that intrathecal administration of very small doses of ifenprodil, just before and ketamine significantly improves its anti-hyperalgesic effect and this association could be useful to control inflammatory pain with less undesirable effects. (c) 2010 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Nucleus Accumbens Dopaminergic Neurotransmission Switches Its Modulatory Action In Chronification Of Inflammatory Hyperalgesia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Dopaminergic neurotransmission in the nucleus accumbens, a central component of the mesolimbic system, has been associated with acute pain modulation. As there is a transition from acute to chronic pain ('chronification'), modulatory structures may be involved in chronic pain development. Thus, this study aimed to elucidate the role of nucleus accumbens dopaminergic neurotransmission in chronification of pain. We used a rat model in which daily subcutaneous injection of prostaglandin E-2 in the hind-paw for 14 days induces a long-lasting state of nociceptor sensitization that lasts for at least 30 days following the end of the treatment. Our findings demonstrated that the increase of dopamine in the nucleus accumbens by local administration of GBR12909 (0.5 nmol/0.25 mu L), a dopamine reuptake inhibitor, blocked prostaglandin E-2-induced acute hyperalgesia. This blockade was prevented by a dopamine D2 receptor antagonist (raclopride, 10 nmol/0.25 mu L) but not changed by a D1 receptor antagonist (SCH23390, 0.5, 3 or 10 nmol/0.25 mu L), both co-administered with GBR12909 in the nucleus accumbens. In contrast, the induction of persistent hyperalgesia was facilitated by continuous infusion of GBR12909 in the nucleus accumbens (0.021 nmol/0.5 mu L/h) over 7 days of prostaglandin E2 treatment. The development of persistent hyperalgesia was impaired by SCH23390 (0.125 nmol/0.5 mu L/h) and raclopride (0.416 nmol/0.5 mu L/h), both administered continuously in the nucleus accumbens over 7 days. Taken together, our data suggest that the chronification of pain involves the plasticity of dopaminergic neurotransmission in the nucleus accumbens, which switches its modulatory role from antinociceptive to pronociceptive.42723802389Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Dietary fatty acid quality affects systemic parameters and promotes prostatitis and pre-neoplastic lesions

Environmental and nutritional factors, including fatty acids (FA), are associated with prostatitis, benign prostate hyperplasia and prostate cancer. We hypothesized that different FA in normolipidic diets (7%) affect prostate physiology, increasing the susceptibility to prostate disorders. Thus, we fed male C57/BL6 mice with normolipidic diets based on linseed oil, soybean oil or lard (varying saturated and unsaturated FA contents and omega-3/omega-6 ratios) for 12 or 32 weeks after weaning and examined structural and functional parameters of the ventral prostate (VP) in the systemic metabolic context. Mongolian gerbils were included because they present a metabolic detour for low water consumption (i.e., oxidize FA to produce metabolic water). A linseed oil-based diet (LO, 67.4% PUFAs, omega-3/omega-6 = 3.70) resulted in a thermogenic profile, while a soybean oil-based diet (SO, 52.7% PUFAs, omega-3/omega-6 = 0.11) increased body growth and adiposity. Mice fed lard (PF, 13.1% PUFA, omega-3/omega-6 = 0.07) depicted a biphasic growth, resulting in decreased adiposity in adulthood. SO and PF resulted in hepatic steatosis and steatohepatitis, respectively. PF and SO increased prostate epithelial volume, and lard resulted in epithelial hyperplasia. Animals in the LO group had smaller prostates with predominant atrophic epithelia and inflammatory loci. Inflammatory cells were frequent in the VP of PF mice (predominantly stromal) and LO mice (predominantly luminal). RNAseq after 12 weeks revealed good predictors of a later-onset inflammation. The transcriptome unveiled ontologies related to ER stress after 32 weeks on PF diets. In conclusion, different FA qualities result in different metabolic phenotypes and differentially impact prostate size, epithelial volume, inflammation and gene expression9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP465699/2014-6sem informação2009/16150-6; 2017/04377-2; 2014/50938-

ATP synthase subunit beta immunostaining is reduced in the sclerotic hippocampus of epilepsy patients

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKA3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected en bloc for HS treatment (n=15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n=10). NKA3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss391149160FAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2013/07559-