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Impact of gestational low-protein intake on embryonic kidney microRNA expression and in the nephron progenitor cells of the male offspring fetus

View ORCID ProfileLetícia de Barros Sene, View ORCID ProfileÍscia Lopes-Cendes, View ORCID ProfileWellerson Rodrigo Scarano, View ORCID ProfileAdriana Zapparoli, View ORCID ProfileJosé Antônio Rocha Gontijo, View ORCID ProfilePatrícia Aline Boer
doi: https://doi.org/10.1101/2020.02.18.954800
Letícia de Barros Sene
1Fetal Programming and Hydroelectrolyte Metabolism Laboratory, Nucleus of Medicine and Experimental Surgery, Department of Internal Medicine, Faculty of Medical Sciences at State University of Campinas, Campinas, SP, Brazil
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  • ORCID record for Letícia de Barros Sene
  • For correspondence: alineboer@yahoo.com.br boer@fcm.unicamp.br gontijo@fcm.unicamp.br lbarrosene@gmail.com
Íscia Lopes-Cendes
2Molecular Genetics Laboratory, Faculty of Medical Sciences at State University of Campinas, Campinas, SP, Brazil
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Wellerson Rodrigo Scarano
3Bioscience Institute, Botucatu, SP, Brazil
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Adriana Zapparoli
1Fetal Programming and Hydroelectrolyte Metabolism Laboratory, Nucleus of Medicine and Experimental Surgery, Department of Internal Medicine, Faculty of Medical Sciences at State University of Campinas, Campinas, SP, Brazil
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José Antônio Rocha Gontijo
1Fetal Programming and Hydroelectrolyte Metabolism Laboratory, Nucleus of Medicine and Experimental Surgery, Department of Internal Medicine, Faculty of Medical Sciences at State University of Campinas, Campinas, SP, Brazil
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  • For correspondence: alineboer@yahoo.com.br boer@fcm.unicamp.br gontijo@fcm.unicamp.br lbarrosene@gmail.com
Patrícia Aline Boer
1Fetal Programming and Hydroelectrolyte Metabolism Laboratory, Nucleus of Medicine and Experimental Surgery, Department of Internal Medicine, Faculty of Medical Sciences at State University of Campinas, Campinas, SP, Brazil
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  • For correspondence: alineboer@yahoo.com.br boer@fcm.unicamp.br gontijo@fcm.unicamp.br lbarrosene@gmail.com
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Abstract

Authors have demonstrated that gestational low-protein (LP) intake offspring showed a lower birth weight, reduced nephrons numbers and renal salt excretion, chronic renal failure, and arterial hypertension development when compared to normal (NP) protein intake group in adult life. The current study was aimed to evaluate the miRNAs and predicted gene expression patterns in the fetal kidney at 17 days of gestational (17-DG) protein-restricted male offspring (LP) in an attempt to elucidate the possible molecular pathways and disorders involved in renal cell proliferation and differentiation during kidney development. By NGS and RT-qPCR were identified, 44 differentially expressed miRNAs, which 19 miRNA were up- and 25 downregulated in LP compared to NP offspring metanephros. Among the top 10 deregulated miRNAs, the study selected 7 miRNAs which its biological targets were related to proliferation, differentiation, and cellular apoptosis processes. As could be seen below, both miRNA-Seq and TaqMan data analysis have shown a consistent change of miRNA expression in LP animals relative to control NP age-matched animals. By immunofluorescence, the LP fetus showed a significant 28% reduction of Six-2 labeled cells when compared to NP offspring associated with a reduced percentage of cell number and c-Myc metanephogenic cap and ureter bud immunostained cells in LP relative to NP offspring.

Additionally, the Ki-67 labeled area in CM was 48% lesser in LP compared to NP age-matched fetus. On the other hand, in LP, the CM and UB β-catenin marked area were 154 and 85% raised, respectively. Mtor immunoreactivity was also significantly higher in LP CM (139%) and UB (104%) compared to the NP fetus. In the LP offspring, the TGFβ-1 in UBs cells staining increased (about 30%). In contrast, Zeb1 metanephros-stained, located in the CM nuclei cells, enhanced 30% in LP and Zeb2 immunofluorescence, although present in whole metanephros structures, was similar in both experimental groups. In conclusion, the present study demonstrates that the miRNAs, mRNAs, and proteins are modified in LP animals with 17 DGs leading to the reduction of the reciprocal induction between CM and UB, and hence the number of nephrons. These findings will facilitate new functional approaches and further studies to elucidate the regulatory mechanisms involved in the processes of proliferation, differentiation, and apoptosis that occurs during renal development.

Footnotes

  • Availability of data and material: Available in https://bv.fapesp.br/pt/pesquisador/671860/leticia-de-barros-sene/ https://repositorio.unesp.br/handle/11449/148594

  • Ethics approval and consent to participate: The Institutional Ethics Committee (CEUA/UNESP, Protocol #446) approved the experimental protocol; the general guidelines established by the Brazilian College of Animal Experimentation were followed throughout the investigation.

  • Competing interest: No conflicts of interest, financial or otherwise, are declared by the authors.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 19, 2020.
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Impact of gestational low-protein intake on embryonic kidney microRNA expression and in the nephron progenitor cells of the male offspring fetus
Letícia de Barros Sene, Íscia Lopes-Cendes, Wellerson Rodrigo Scarano, Adriana Zapparoli, José Antônio Rocha Gontijo, Patrícia Aline Boer
bioRxiv 2020.02.18.954800; doi: https://doi.org/10.1101/2020.02.18.954800
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Impact of gestational low-protein intake on embryonic kidney microRNA expression and in the nephron progenitor cells of the male offspring fetus
Letícia de Barros Sene, Íscia Lopes-Cendes, Wellerson Rodrigo Scarano, Adriana Zapparoli, José Antônio Rocha Gontijo, Patrícia Aline Boer
bioRxiv 2020.02.18.954800; doi: https://doi.org/10.1101/2020.02.18.954800

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