Impact of maternal protein restriction on Hypoxia-Inducible Factor (HIF) expression in male fetal kidney development.

BACKGROUND: Kidney developmental studies have demonstrated molecular pathway changes that may be related to decreased nephron numbers in the male 17 gestational days (17GD) low protein (LP) intake offspring compared to normal protein intake (NP) progeny. Here, we evaluated the HIF-1 and components of its pathway in the kidneys of 17-GD LP offspring to elucidate the molecular modulations during nephrogenesis. METHODS: Pregnant Wistar rats were allocated into two groups: NP (regular protein diet-17%) or LP (Low protein diet-6%). Taking into account miRNA transcriptome sequencing previous study (miRNA-Seq) in 17GD male offspring kidneys investigated predicted target genes and proteins related to the HIF-1 pathway by RT-qPCR and immunohistochemistry. RESULTS: In the present study, in male 17-GD LP offspring, an increased elF4, HSP90, p53, p300, NFκß, and AT2 gene encoding compared to the NP progeny. Higher labeling of HIF-1α CAP cells in 17-DG LP offspring was associated with reduced elF4 and phosphorylated elF4 immunoreactivity in LP progeny CAP cells. In 17DG LP, the NFκß and HSP90 immunoreactivity was enhanced, particularly in the CAP area. DISCUSSION AND CONCLUSION: The current study supported that the programmed reduced nephron number in the 17-DG LP offspring may be related to changes in the HIF-1α signaling pathway. Factors that facilitate the transposition of HIF-1α to progenitor renal cell nuclei, such as increased NOS, Ep300, and HSP90 expression, may have a crucial role in this regulatory system. Also, HIF-1α changes could be associated with reduced transcription of elF-4 and its respective signaling path.

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

BACKGROUND: The kidney ontogenesis is the most structurally affected by gestational protein restriction, reducing 28% of their functional units. The reduced nephron number is predictive of hypertension and cardiovascular dysfunctions that are generally observed in the adult age of most fetal programming models. We demonstrate miRNAs and predict molecular pathway changes associated with reduced reciprocal interaction between metanephros cap (CM) and ureter bud (UB) and a 28% decreased nephron stem cells in the 17 gestational days (17GD) low protein (LP) intake male fetal kidney. Here, we evaluated the same miRNAs and predicted targets in the kidneys of 21GD and at 7 days of life (7DL) LP offspring to elucidate the molecular modulations during nephrogenesis. METHODS: Pregnant Wistar rats were allocated into two groups: NP (regular protein diet- 17%) or LP (diet-6%). miRNA transcriptome sequencing (miRNA-Seq) was performed on the MiSeq platform from 21GD and 7DL male offspring kidneys using previously described methods. Among the top 10 dysfunctional regulated miRNAs, we validated 7 related to proliferation, differentiation, and apoptosis processes and investigated predicted target genes and proteins by RT-qPCR and immunohistochemistry. RESULTS: In 21GD, LP fetuses were identified alongside 21 differently expressed miRNAs, of which 12 were upregulated and 9 downregulated compared to age-matched NP offspring. In 7-DL LP offspring, the differentially expressed miRNAs were counted to be 74, of which 46 were upregulated and 28 downregulated. The curve from 17-GD to 7-DL shows that mTOR was fundamental in reducing the number of nephrons in fetal kidneys where the mothers were subjected to a protein restriction. IGF1 and TGFß curves also seemed to present the same mTOR pattern and were modulated by miRNAs 181a-5p, 181a-3p, and 199a-5p. The miRNA 181c-3p modulated SIX2 and Notch1 reduction in 7-DL but not in terms of the enhanced expression of both in the 21-GD, suggesting the participation of an additional regulator. We found enhanced Bax in 21-GD; it was regulated by miRNA 298-5p, and Bcl2 and Caspase-3 were controlled by miRNA (by 7a-5p and not by the predicted 181a-5p). The miRNA 144-3p regulated BCL6, which was enhanced, as well as Zeb 1 and 2 induced by BCL6. These results revealed that in 21GD, the compensatory mechanisms in LP kidneys led to the activation of UB ramification. Besides, an increase of 32% in the CM stem cells and a possible cell cycle halt of renal progenitor cells, which remaining undifferentiated, were observed. In the 7DL, much more altered miRNA expression was found in LP kidneys, and this was probably due to an increased maternal diet content. Additionally, we verified the activation of pathways related to differentiation and consumption of progenitor cells.

Gestational and Breastfeeding Low-Protein Intake on Blood Pressure, Kidney Structure, and Renal Function in Male Rat Offspring in Adulthood.

Background: Our previous studies demonstrated that maternal protein-restricted (low-protein, LP) 16-week-old offspring had pronounced nephron number reduction and arterial hypertension associated with an unchanged glomerular filtration rate (GFR). An enhanced gomerular area may be related to increased glomerular filtration and overflow, which accounts for glomerular filtration barrier breakdown and early glomerulosclerosis. The effect of protein restriction during gestational and breastfeeding periods is unknown. Method: The functional e-structural kidney evaluation was obtained using lithium and creatinine clearance, kidney morphometry, immunoblotting, and immunostaining analysis in 16 and 24-week-old LP offspring compared to age-matched NP progeny. Results: Low protein rats' progeny had significantly reduced birth weight, without previous catch-up growth phenomena, in parallel with a decreased adiposity index. Transforming growth factor-beta 1 (TGF-ß1) glomerular expression was significantly enhanced in the LP group. Also, the LP offspring had a 38% lower nephron number and an increased glomerular volume. They also presented with a higher cardiac index and arterial blood pressure compared with age-matched NP offspring. The LP rats exhibited augmented Na+/K+-ATPase in the proximal segments, and NOS1 immunoreactivity in whole renal tissue was associated with sodium retention in the proximal nephron segments. We also found significantly enhanced collagen content associated with increased TGFß1 and ZEB1/2 renal immunoreactivity in LP offspring compared with NP offspring. Increased hypertrophy markers in LP podocytes were associated with an amplified IL-6/STAT3 pathway activity. Conclusion: To our knowledge, these are the first data demonstrating renal functional and structural changes in protein restriction during gestation and lactation model of fetal programming. The fetal-programmed adult offspring showed pronounced structural glomerular disorders with an accentuated and advanced fibrosis stage, without a change in the GFR. These findings suggest that the glomerular enhanced TGF-ß1 action may induce ZEB1/2 expression that may cause glomeruli epithelial-to-mesenchymal transition. Besides, decreased nephron number in the LP offspring with preserved glomerular function may be related to protective or even attenuate the activated IL-6/STAT3 pathway.