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MRNAs, miRNAs, and regulatory protein expression nephrogenesis in rats programmed by protein restriction in utero

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Letícia de Barros Sene
Total Authors: 1
Document type: Doctoral Thesis
Press: Botucatu. 2017-01-25.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
Defense date:
Advisor: Patrícia Aline Boer; Wellerson Rodrigo Scarano

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. (AU)

FAPESP's process: 12/18492-4 - Comparative study of the effects of protein restriction in utero and dexamethasone in vitro exposure during nephrogenesis: assessment of gene expression, of miRNAs, protein, and morphometric in metanephric mesenchyme
Grantee:Letícia de Barros Sene
Support type: Scholarships in Brazil - Doctorate