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

PLoS One. 2021 Feb 5;16(2):e0246289. doi: 10.1371/journal.pone.0246289. eCollection 2021.


Background: Here, we have demonstrated that gestational low-protein (LP) intake offspring present lower birth weight, reduced nephron numbers, renal salt excretion, arterial hypertension, and renal failure development compared to regular protein (NP) intake rats in adulthood. We evaluated the expression of various miRNAs and predicted target genes in the kidney in gestational 17-days LP (DG-17) fetal metanephros to identify molecular pathways involved in the proliferation and differentiation of renal embryonic or fetal cells.

Methods: Pregnant Wistar rats were classified into two groups based on protein supply during pregnancy: NP (regular protein diet, 17%) or LP diet (6%). Renal miRNA sequencing (miRNA-Seq) performed on the MiSeq platform, RT-qPCR of predicted target genes, immunohistochemistry, and morphological analysis of 17-DG NP and LP offspring were performed using previously described methods.

Results: A total of 44 miRNAs, of which 19 were up and 25 downregulated, were identified in 17-DG LP fetuses compared to age-matched NP offspring. We selected 7 miRNAs involved in proliferation, differentiation, and cellular apoptosis. Our findings revealed reduced cell number and Six-2 and c-Myc immunoreactivity in metanephros cap (CM) and ureter bud (UB) in 17-DG LP fetuses. Ki-67 immunoreactivity in CM was 48% lesser in LP compared to age-matched NP fetuses. Conversely, in LP CM and UB, β-catenin was 154%, and 85% increased, respectively. Furthermore, mTOR immunoreactivity was higher in LP CM (139%) and UB (104%) compared to that in NP offspring. TGFβ-1 positive cells in the UB increased by approximately 30% in the LP offspring. Moreover, ZEB1 metanephros-stained cells increased by 30% in the LP offspring. ZEB2 immunofluorescence, although present in the entire metanephros, was similar in both experimental groups.

Conclusions: Maternal protein restriction changes the expression of miRNAs, mRNAs, and proteins involved in proliferation, differentiation, and apoptosis during renal development. Renal ontogenic dysfunction, caused by maternal protein restriction, promotes reduced reciprocal interaction between CM and UB; consequently, a programmed and expressive decrease in nephron number occurs in the fetus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Diet, Protein-Restricted / adverse effects*
  • Female
  • Kidney / embryology*
  • Kidney / metabolism
  • Male
  • Maternal Nutritional Physiological Phenomena*
  • MicroRNAs / metabolism*
  • Nephrons / embryology*
  • Nephrons / metabolism
  • Pregnancy
  • Rats
  • Rats, Wistar
  • Real-Time Polymerase Chain Reaction
  • Stem Cells / metabolism*


  • MicroRNAs

Grant support

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2013/12486-5 and 2014/50938-8), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 465699/2014-6).