Fetal 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.
Department of Structural and Functional Biology, Bioscience Institute, São Paulo State University, Botucatu, SP, Brazil.
PLoS One. 2021 Feb 5;16(2):e0246289. doi: 10.1371/journal.pone.0246289. eCollection 2021.
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.
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.
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.
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.
在这里，我们已经证明，与正常蛋白质（NP）摄入的大鼠相比，妊娠期低蛋白（LP）摄入的后代在成年时体重较低、肾单位数量减少、盐排泄减少、动脉高血压和肾衰竭发展。我们评估了妊娠 17 天 LP（DG-17）胎儿后肾间充质中各种 miRNA 的表达，并预测了靶基因，以鉴定参与肾胚胎或胎儿细胞增殖和分化的分子途径。
根据妊娠期间蛋白质供应情况，将怀孕的 Wistar 大鼠分为两组：NP（正常蛋白质饮食，17%）或 LP 饮食（6%）。在 MiSeq 平台上进行肾 miRNA 测序（miRNA-Seq），对预测的靶基因进行 RT-qPCR，使用以前描述的方法对 17-DG NP 和 LP 后代进行免疫组织化学和形态学分析。
与年龄匹配的 NP 后代相比，17-DG LP 胎鼠中总共鉴定出 44 种 miRNA，其中 19 种上调，25 种下调。我们选择了 7 种参与增殖、分化和细胞凋亡的 miRNA。我们的研究结果表明，17-DG LP 胎鼠的后肾间充质帽（CM）和输尿管芽（UB）中的细胞数量减少，Six-2 和 c-Myc 免疫反应性降低。与年龄匹配的 NP 胎鼠相比，LP 胎鼠的 CM 中 Ki-67 免疫反应性降低了 48%。相反，在 LP CM 和 UB 中，β-连环蛋白分别增加了 154%和 85%。此外，LP CM（139%）和 UB（104%）中 mTOR 免疫反应性高于 NP 后代。LP 后代的 UB 中 TGFβ-1 阳性细胞增加了约 30%。此外，LP 后代的肾间充质中 ZEB1 染色细胞增加了 30%。尽管 LP 后代的整个肾间充质中都存在 ZEB2 免疫荧光，但在两个实验组中都相似。
母体蛋白质限制改变了肾脏发育过程中参与增殖、分化和凋亡的 miRNA、mRNA 和蛋白质的表达。母体蛋白质限制引起的肾脏发生障碍促进了 CM 和 UB 之间的相互作用减少，因此胎儿肾单位数量减少。
