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

Abstract

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.

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

“…During kidney development in LP 17GD offspring, we have demonstrated that miR-181c enhanced the expression of Six2 and negatively regulates cell proliferation (Sene et al, 2021), confirming results by Lv et al (2014).…”

“…At 21GD, PCNA and c-myc mRNA expression was downregulated (Figure 4) in maternal protein-restricted offspring. Using results obtained in the 17GD metanephros using the same model presented here (Sene et al, 2021) and 21GD and 7DL kidneys, we have traced curves where we can observe the more detailed and comparative view of mRNAs expression control by microRNAs (Figure 4).…”

“…The low nephron number is related to hypertension, and in hypertensive patients, approximately 46% of the number of nephrons is reduced (Mackenzie et al, 1996). Prior experimental studies from our lab and other authors have demonstrated lower birth weight, 28% fewer nephrons, reduced renal salt excretion, chronic renal failure, and enhanced systolic pressure from 8 to 16 weeks of life in gestational low-protein (LP) intake compared to standard (NP) protein intake offspring in adulthood (Schreuder et al, 2006;Mesquita et al, 2010a,b;Sene et al, 2013Sene et al, , 2018Sene et al, , 2021. However, information regarding the molecular mechanisms of the etiopathogenesis of nephrogenesis cessation is still scarce.…”

“…Fetal programming is characterized as any psychological and nutritional stress during development, which leads to long-term effects on kidney structure and function disorders with an increasing predictive chance to develop chronic renal disease ( Lucas, 1998 ). Disturbance in fetal programming results in low birth weight, fewer nephrons, and increased risk of cardiovascular and renal disorders in adulthood ( Mesquita et al, 2010a , b ; Sene et al, 2013 , 2018 , 2021 ). The low nephron number is related to hypertension, and in hypertensive patients, approximately 46% of the number of nephrons is reduced ( Mackenzie et al, 1996 ).…”

“…We recently demonstrated in the fetus at 17 days of gestation (GD) protein-restricted male fetus changes in metanephros miRNAs and predicted mRNA expression that encodes proteins related to nephrogenesis a 28% reduction in nephrogenic stem cells in the cap metanephric (CM). We suggested that miRNAs, mRNAs, and protein disruption could have reduced proliferation and promote early cell differentiation ( Sene et al, 2021 ). The rats are remaining the nephrogenesis after birth.…”