Expressão de mRNAs, miRNAS e proteínas reguladoras da nefrogênese em ratos programados por restrição proteica in utero
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Data
2016-12-20
Autores
Sene, Letícia de Barros [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Tanto a desnutrição proteico-calórica quanto a hipertensão representam problemas globais de saúde pública. Estudos epidemiológicos em diversas populações, bem como resultados experimentais indicam que as condições nutricionais intrauterinas "programam" o desenvolvimento de hipertensão e doença cardíaca coronariana em adultos. A privação de proteína, durante o período ativo de nefrogênese, provoca redução do número de néfrons no momento do nascimento. Assim, a predisposição para hipertensão arterial pode ser determinada, pelo menos em parte, pelo desenvolvimento anormal dos rins. No entanto, não é conhecido os mecanismos que comprometem o processo de nefrogênese. Estudos recentes mostram que o número de células-tronco presentes no cap mesenquimal (CM), em torno da extremidade dos brotos do ureter, determina o número de néfrons que serão formados. Nosso grupo demonstrou anteriormente, redução significativa (27%) do número de néfrons, em prole masculina adulta, proveniente de ratas submetidas à restrição proteica gestacional. O objetivo deste trabalho foi verificar a área, o índice de morte e proliferação celular, a expressão gênica e de miRNAs, número de células positivas para proteínas controladoras do ciclo celular e marcadoras de células tronco progenitoras em metanefros de machos da prole de ratas submetidas à restrição proteica gestacional, comparativamente aos seus controles, em diferentes períodos da ontogênese renal. Ratas Wistar foram alimentadas durante a prenhez com dietas com conteúdo normal de proteína (NP 17% de caseína) ou baixo conteúdo de proteína (LP 6% de caseína). No 17º e 21º de dia de gestação (DG) e no 7º dia pós-natal (DPN) os rins foram processados para coloração de HE, imunolocalização, sequenciamento (NGS) e RT-qPCR. Foi obtido um grande número de miRNAs diferencialmente expressos, entre eles, o miR-127-3p, let-7a-5p, miR-199a-5p, miR-298-5p, miR-144-3p, miR-181a-5p e o miR-181c-5p, que foram validados por RT-qPCR. Os animais LP, com 17 DG, apresentaram redução da área renal total, da área nefrogênica e redução de células em proliferação. Os animais LP, com 21DG, também apresentaram redução da área nefrogênica, além de menor peso do rim. O miR-127-3p, o miR-144-3p e o miR-199a-5p está menos expresso nos animais LP de 17 DG, enquanto que o miR-181a-5p, miR-181c-3p e o let-7a-5p estão mais expressos. O Six2, está diminuído nos animais LP de 17 DG, assim como o c-Myc, Vegf e Notch1. Já Bax, Tgfb1, Bcl6, c-Ret, Map2k2, Mki67, mTOR, β-Catenina, Zeb1, Zeb2 e Igf1 estão mais expressos nesses animais. Já nos animais LP de 21 DG o miR-127-3p, miR-298-5p, let-7a-5p, miR-181a-5p e o miR-181c-3p estão com sua expressão aumentada. Six2, Bax, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, c-Ret, Prdm1, Vegf, Mki67, β-Catenina, Zeb1, Zeb2, Notch1 e Igf1 estão mais expressos nos animais LP de 21 DG. Enquanto que Pcna, c-Myc estão com sua expressão reduzida. A expressão do miR-181a-5p está maior nos animais LP com 7 DPN e a expressão de let-7a-5p está reduzida. mTOR está regulada negativamente nos animais LP de 7 DPN, e Bax, Bim, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, Ciclina A, Map2k2, Prdm1, Vegf, β-Catenina, Zeb1, Zeb2, Notch1 e Igf1 estão regulados positivamente. Em conclusão, este estudo indicou que a redução do número de néfrons (28%) induzida por restrição proteica gestacional é determinada, pelo menos em parte, pela redução na população de células tronco progenitoras (28%) e de mitose no 17º dia gestacional. A restrição proteica gestacional altera a expressão de miRNAs e genes-alvos que estão associados com processos de apoptose, proliferação e diferenciação celular que são essenciais durante o desenvolvimento renal.
Both the protein-calorie malnutrition and hypertension represent global problems of public health. Epidemiologic studies in diverse populations, as well as experimental results indicate that intrauterine nutritional conditions "program" the development of hypertension and coronary heart disease in adults. The protein deprivation, during the active period of nephrogenesis, causes a reduction in the number of nephrons at birth. Thus, the predisposition to hypertension can be determined, at least in part, by abnormal kidney development. However, it is not known the mechanism that lead to undertakes the nephrogenesis process. Recent studies show that the number of stem cells present in the mesenchymal cap (CM), around the tip of the bud of the ureter, determines the number of nephrons to be formed. We have demonstrated, in adult male offspring, from rats submitted to gestational protein restriction, a significant reduction (27%) of the nephron number. The aim of present study was investigated to verify the area, cell death and proliferation index, gene and miRNA expression, number of cells positive for cell cycle control proteins and markers of progenitor stem cells in metanephro male offspring of rats submitted to gestational protein restriction, compared to their controls, in different periods of renal ontogenesis. Wistar rats were fed during pregnancy with normal-protein (NP 17% casein) or low-protein (LP 6% casein) diet. In the 17th and 21th gestational day (DG) and 7th day of life (DPN) the kidneys were processed to immunolocalization, Next Sequencing Generation (NGS), and RT-qPCR. A big number of differentially expressed miRNAs was obtained among them, miR-127-3p, let-7a-5p, miR-199a-5p, miR-298-5p, miR-144-3p, miR-181a-5p and miR-181c-5p, these were validated by RT-qPCR. LP animals, with 17 DG, presented reduction of the total renal area, the nephrogenic area and cells proliferating were reduced. LP animals, with 21DG, also presented a reduction of the nephrogenic area, as well as a lower weight of the kidney. miR-127-3p, miR-144-3p and miR-199a-5p were downregulated in the 17DG-LP animals, while miR-181a-5p, miR-181c-3p and let-7a- 5p were upregulated. Six2, was decreased in 17 DG-LP animals, as well as c-Myc, Vegf and Notch1. Bax, Tgfb1, Bcl6, c-Ret, Map2k2, Mki67, mTOR, β-Catenin, Zeb1, Zeb2 and Igf1 were upregulated in these animals. In the 21 DG-LP animals, miR-127-3p, miR-298-5p, let-7a-5p, miR-181a-5p and miR-181c-3p were increased expression. Six2, Bax, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, c-Ret, Prdm1, Vegf, Mki67, β-Catenina, Zeb1, Zeb2, Notch1 e Igf1 were more expressed in 21DG-LP animals. Whereas Pcna, c-Myc are reduced in expression. Expression of miR-181a-5p was higher in LP animals with 7 DPN and let-7a-5p expression was reduced. mTOR was downregulated in the LP animals of 7 DPN, and Bax, Bim, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, Cyclin A, Map2k2, Prdm1, Vegf, β-Catenin, Zeb1, Zeb2, Notch1 and Igf1 are regulated positively. In conclusion, this study indicated that the nephron number reduction (28%) induced by gestational protein restriction is determined, at least in part, by reduction in mitosis and kidney mesenchymal stem/progenitor cell population (28%) at 17th gestational day. Gestational protein restriction alters the expression of miRNAs and target genes that are associated with proliferation, cell differentiation and apoptosis processes that are essential during renal development.
Both the protein-calorie malnutrition and hypertension represent global problems of public health. Epidemiologic studies in diverse populations, as well as experimental results indicate that intrauterine nutritional conditions "program" the development of hypertension and coronary heart disease in adults. The protein deprivation, during the active period of nephrogenesis, causes a reduction in the number of nephrons at birth. Thus, the predisposition to hypertension can be determined, at least in part, by abnormal kidney development. However, it is not known the mechanism that lead to undertakes the nephrogenesis process. Recent studies show that the number of stem cells present in the mesenchymal cap (CM), around the tip of the bud of the ureter, determines the number of nephrons to be formed. We have demonstrated, in adult male offspring, from rats submitted to gestational protein restriction, a significant reduction (27%) of the nephron number. The aim of present study was investigated to verify the area, cell death and proliferation index, gene and miRNA expression, number of cells positive for cell cycle control proteins and markers of progenitor stem cells in metanephro male offspring of rats submitted to gestational protein restriction, compared to their controls, in different periods of renal ontogenesis. Wistar rats were fed during pregnancy with normal-protein (NP 17% casein) or low-protein (LP 6% casein) diet. In the 17th and 21th gestational day (DG) and 7th day of life (DPN) the kidneys were processed to immunolocalization, Next Sequencing Generation (NGS), and RT-qPCR. A big number of differentially expressed miRNAs was obtained among them, miR-127-3p, let-7a-5p, miR-199a-5p, miR-298-5p, miR-144-3p, miR-181a-5p and miR-181c-5p, these were validated by RT-qPCR. LP animals, with 17 DG, presented reduction of the total renal area, the nephrogenic area and cells proliferating were reduced. LP animals, with 21DG, also presented a reduction of the nephrogenic area, as well as a lower weight of the kidney. miR-127-3p, miR-144-3p and miR-199a-5p were downregulated in the 17DG-LP animals, while miR-181a-5p, miR-181c-3p and let-7a- 5p were upregulated. Six2, was decreased in 17 DG-LP animals, as well as c-Myc, Vegf and Notch1. Bax, Tgfb1, Bcl6, c-Ret, Map2k2, Mki67, mTOR, β-Catenin, Zeb1, Zeb2 and Igf1 were upregulated in these animals. In the 21 DG-LP animals, miR-127-3p, miR-298-5p, let-7a-5p, miR-181a-5p and miR-181c-3p were increased expression. Six2, Bax, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, c-Ret, Prdm1, Vegf, Mki67, β-Catenina, Zeb1, Zeb2, Notch1 e Igf1 were more expressed in 21DG-LP animals. Whereas Pcna, c-Myc are reduced in expression. Expression of miR-181a-5p was higher in LP animals with 7 DPN and let-7a-5p expression was reduced. mTOR was downregulated in the LP animals of 7 DPN, and Bax, Bim, Casp3, Col1, Gdnf, Tgfb1, Bcl2, Bcl6, Cyclin A, Map2k2, Prdm1, Vegf, β-Catenin, Zeb1, Zeb2, Notch1 and Igf1 are regulated positively. In conclusion, this study indicated that the nephron number reduction (28%) induced by gestational protein restriction is determined, at least in part, by reduction in mitosis and kidney mesenchymal stem/progenitor cell population (28%) at 17th gestational day. Gestational protein restriction alters the expression of miRNAs and target genes that are associated with proliferation, cell differentiation and apoptosis processes that are essential during renal development.
Descrição
Palavras-chave
Cap mesenquimal, Néfrons, Rins - Desenvolvimento, Feto - Desenvolvimento, Dieta com restrição de proteínas