The understanding of the cell and nephrogenesis molecular mechanisms has overgrown in late years (Cerqueira et al., 2017; Costantini and Kopan, 2010; Hendry et al., 2011; Kopan et al., 2014; Little and McMahon, 2012). However, many regulatory factors and signaling pathways involved in renal ontogenesis maintain unclear (Wang et al., 2018). It is known that miRNAs play a crucial role in regulating gene expression during the renal developmental processes (Lv et al., 2014; Marrone and Ho, 2014; Phua et al., 2015b; Wei et al., 2014). So far, to our knowledge, no prior study has performed an integrative miRNA and mRNA expression profiling analysis in maternal low-protein intake 17-DG male metanephros.
In the current study has proposed a novel molecular pathway that may be involved in the early nephrogenesis interruption and, consequently, reducing the number of nephrons. The present study, by NGS, 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 and specific change of miRNA expression in LP animals relative to control NP age-matched animals.
The miR-181 family is composed of four highly conservative members, miR-181a, miR-181b, miR-181c, and miR-181d (Ji et al., 2010). In neoplastic cells, miR-181a plays the role of the tumor suppressor gene, inhibiting the proliferation and cell migration and, inducing cellular apoptosis (Chen. et al., 2010). In the current study, increased expression of miR-181a-5p in 17-DG LP relative to age-matched NP offspring; also, here, was demonstrated a 2-fold enhanced Bax/Bcl2 mRNA ratio in LP compared to NP offspring. These findings reaffirm increased CM apoptosis activity, although caspase mRNA expression is not altered, which may indicate a post-transcriptional mechanism on apoptosis regulation. Previous studies have shown that the BCL family promotes cytochrome release from mitochondria, and then inhibit the activation of Casp3 and subsequent cellular apoptosis (Scorrano and Korsmeyer, 2003). Li and colleagues, in an acute lung injury model, have shown that overexpressed miR-181a is related to decreased Bcl2 protein level; conversely, the inhibition of miR-181a increase Bcl2 levels (Li et al., 2016). The current study confirming Lv et al. (2014) demonstrated that miR-181c regulates negatively, the expression of Six2 expression and cell proliferation, in parallel to the loss of mesenchymal cells phenotype during kidney development in LP 17-DG offspring.
Studies from Xiang et al. demonstrate that miR-144 expression was decreased in kidney carcinoma cells (Xiang et al., 2016). On the other hand, they also showed that enhanced miR-144 expression suppresses renal carcinoma proliferation and decreasing the G2/M phase cells ratio. Our results show reduced expression of miR-144-3p in 17-DG LP offspring CAP, suggesting that it may be inducing cell proliferation.
Additionally, in renal cancer, the mTOR pathway plays an important pathogenic role in proliferation and response apoptosis during tumor development (Bailey et al., 2014; Ribback et al., 2015). Moreover, another study was demonstrated that the mTOR signaling pathway is crucial for the decreased number of nephrons in fetal kidneys whose mothers were subjected to the nutrient restriction (Nijland et al., 2007). The Xiang group demonstrated a significant molecular correlation between miR-144 and mTOR. They observed that overexpression of miR-144 inhibits the mTOR gene and protein expression. Conversely, the lower expression of miR-144 enhances mTOR mRNA expression (Xiang et al., 2016). Here, LP animals demonstrated an increased expression of mTOR. This finding suggests that the miR-144 expression may regulate mTOR, but not only by it, to promote cell proliferation.
The mammalian target of rapamycin complex 1 (mTORC1) is known to be indispensable for healthy embryonic development, has how to function to regulate the balance between growth and autophagy in physiological conditions and environmental stress (Gürke et al., 2016). It has been suggested that mTOR signaling plays a central role in the perception and response to intracellular nutrient availability (Xiang et al., 2016). Thus, this pathway would undoubtedly be involved in cellular responses in maternal protein underfeeding animals. The previous study demonstrated that an increase in mTOR was fundamental in reducing the number of nephrons in fetal kidneys, whose mothers were subjected to a nutrient restriction (Nijland et al., 2007). However, Volovelsky et al. (2018) reported that conditional deletion of mTOR in nephron progenitor cells profoundly disrupted nephrogenesis, and hemizygous deletion led to a significant reduction in nephron endowment. In the present study, the mRNA expression for mTOR in 17-DG LP offspring was remarkably increased, and the protein immunoreactivity was also enhanced, respectively, 139% and 104% in CM cells and UB.
The miR-127 has an essential role in proliferation, differentiation, and development, and one of its targets the proto-oncogene Bcl6 (Chen et al., 2013). Pan et al., show that in liver cells, that miR-127 underexpression is related to increased cell proliferation, and its overexpression was associated with proliferation inhibition (Pan et al., 2012). Also, Chen and col. classified this miRNA as a new regulator of cell senescence via Bcl6 (Chen et al., 2013). Increased mRNA accompanied the reduction of miR 127-3p in 17-DG LP offspring for Ki67 associated with an increase of Bcl-6 in CM.
In this study, we also observed that the animals from the LP group presented a reduction in the nephrogenic area, corroborating the findings of Menendez-Castro and col. (2013 and 2014), who observed that both proliferative activity and the nephrogenic zone were significantly reduced in animals that suffered protein restriction (8.4% casein) relative to the controls group. We also observed a significant reduction of cells positively labeled for Ki67 in CM in protein-restricted animals.
The study showed that overexpression of miR-199a-5p reduces cystic cell proliferation and induces apoptosis, in addition to controlling the cell cycle (Sun et al., 2015). Expression of miR-199a-5p is reduced in LP, and the transcription of Ki67 and Map2k2 are increased. These findings indicate that gestational undernutrition promotes differentiation in detriment of proliferation associated with increased Zeb2 mRNA expression. Zeb2 is a transcription factor that regulates the renal tubule morphogenesis acting on proximal tubule development and glomerular-tubular junction (Rasouly et al., 2017).
The let-7 miRNA family expression has been extensively studied in several fetal tissues and, priority is related with reduced proliferation and induced cell differentiation (Ambros, 2012; Bao et al., 2013; Copley and Eaves, 2013; Meza-Sosa et al., 2014; Nagalakshmi et al., 2015). It has been shown in higher organisms, enhanced let-7 levels during embryogenesis (Schulman et al., 2005), and also that let-7a mature form is upregulated during the developmental mouse brain (Wulczyn et al., 2007). A study from Nagalakshmi et al. (2015) has shown that let-7 miRNAs potentially upregulated UB epithelial cells fate from precursor to the differentiated state. On the other hand, Yermalovich et al. (2019) have demonstrated the overexpression of Lin28b, an RNA-binding protein, is associated with suppressive let-7 miRNAs expression and elongated nephrogenesis, via the let-7 miRNAs upregulation. Thus, in the current study, the enhanced let-7a-5p miRNA expression in LP offspring might be associated with reducing CM cell proliferation, compromising the whole nephrogenesis relative to the NP group. While the heterochronic genes lin28 and let-7 are well-established regulators of developmental timing in invertebrates, their role in mammalian organogenesis was not fully understood. In the present study, we unprecedently report the reciprocal Lin28b/let-7 axis relationship during fetus kidney development in LP rats. So, we may hypothesize that overexpressed let-7 miRNAs, direct or indirectly, throughout a transient reduced LIN28B, might decrease the nephrogenesis process, and consequently, the kidney functional unit numbers potentially via upregulation of the Igf2/H19 locus. Conversely, a supposed kidney-specific regular expression of Lin28b normalizes whole kidney development, as was suggested in this study.
Here, such as in previous studies, was also observed increased let-7a-5p miRNA in parallel to the fall of c-Myc expression (Chang et al., 2008; Sampson et al., 2007). Prior study has demonstrated the involvement of MYC proteins in proliferation, growth, apoptosis and cell differentiation, critical events during renal organogenesis (Couillard and Trudel, 2009). In the LP 17-DG offspring, metanephric c-myc gene expression was reduced, and the CM c-myc immunoreactivity area was either 14% smaller, both when compared to NP offspring. Coincidently, CM cell number was also 14% reduced in LP, and KI-67 immunoreactivity was decreased even (48%) in LP relative to the NP group. Sustaining the present findings, authors have shown that c-myc plays a role in the developmental process, being crucial in the final phase of the UB branching by modulation of CM progenitor cell proliferation (Couillard and Trudel, 2009). It seems that let-7 should be strongly expressed at specific late stages of differentiation, but keeps downregulated in stem cells maintaining these cells in an undifferentiated state. Thus, the current study established that the let-7 family of miRNAs promotes MYC expression by a transcriptionally induced let-7 repressor, LIN28 enhancement and, post-transcriptional expressed LIN28 RNA binding-protein, promoting downregulated upon LP kidney cells differentiation (Fig. 10).
The previous study has demonstrated that Six2 gene expression is downregulated during fetal ontogenesis when associated with reduced numbers of nephrons, arterial hypertension, and chronic renal failure in adulthood (Fogelgren et al., 2009). In parallel, the present study shows a significant 28% reduced Six2 positive cells in 17-DG LP compared to NP offspring, in similar proportion to previously observed decreased nephrons numbers in restricted-protein offspring. Six2, a specific marker of the renal stem cell system, is required to maintain under control the fate cell and the nephron progenitor cells population by suppressing signals induced-epithelial differentiation (Herzlinger et al., 1992). Its expression maintains a pool of CM cells available in the undifferentiated stage during renal development (Oliver et al., 1995; Self et al., 2006; Humphreys et al., 2008). The present data is sustained by a prior study in c-myc transgenic mice kidneys (Couillard and Trudel, 2009), that showed a decreased c-myc and six-2 immunopositive CM cells associated with reduced CM stem cell proliferation in 17-DG LP offspring.
Notch signaling is involved in the negative regulation of Six2, suggesting profound Notch impact on the gene regulatory network on the maintenance of nephron progenitor cells (Cheng, 2003; Cheng et al., 2009). Once that reduced expression of Six2 is required for differentiation of the nephron progenitors, the Notch signaling is fundamental to downregulate Six2. Thus, we may argue that reduced c-myc and Notch signaling in this study might stimulate renal cell differentiation since it regulates the size of the CM cell population. In several cases, the Wnt/β-catenin pathway initiates the differentiation of nephron progenitor cells being, however, essential for the maintenance of nephron progenitor cells (Brown et al., 2015). Thus, a low level of β-catenin might be required for the maintenance of Six2 expression, and a high level of β-catenin may contribute to the downregulation of Six2. Wnt/β-catenin and Notch signals may coordinate that Six2 expression regulation.
Additionally, elevated levels of β-catenin activity also determine nephron progenitor cell fate. Usually, only when a sufficient pool of CM cells is reached, nephrogenesis proceed. In this way, we assume that 17-DG LP offspring has a 28% reduction of CM, which will affect the percentage of nephrons in adulthood. Thus, partial conclusions of the present study sustain that, in LP offspring metanephros, the increased let-7a-5p and β-catenin expression and reduced Notch signal modulate the c-myc, six2, and KI-67 leading to reduction of progenitor cells self-renewal. By the way, the exhaustion of the remaining CM progenitor cell endowment, in turn, predisposes to reduced nephron numbers, arterial hypertension, and renal disorders in adult age (Fig. 10).
As previously observed in our Lab, a reduction of 28.3% in ureteric bud branches after 14.5 days of gestational protein restriction (Mesquita et al., 2010). Genes encoding the peptide growth factor glial-derived neurotrophic factor (GDNF), c-Ret tyrosine kinase receptor, and its coreceptor Gfra1 are recognized as crucial regulators of ureteric bud outgrowth (Davis et al., 2015). Thus, the c-ret receptor, when activated by GDNF, induces UB branching. In the 17-DG offspring, was observed a significant increase in c-Ret receptor coding mRNA, which would theoretically lead to a rise in UB ramifications. However, in the present study, the GDNF expression was unchanged, being plausible to suppose that, despite the increase of c-Ret, the UB branching did not also change. How is it known that Six2 regulates transcription of GDNF (Brodbeck et al., 2004), thus, the reduction of 28% in the cells positive for Six2 could affect, in the same proportion the GDNF expression which in turn, would act in the decrease of 28.3% of the ramifications of the ureteric bud as previously observed (Mesquita et al., 2010).
Karner et al. (2011) demonstrated that the signaling pathway is Wnt9b/ β-catenin is required both for nephron progenitor cell renewal and for differentiation, thus being essential for the formation of nephrons during embryogenesis. However, it is not yet known how β-catenin regulates these two processes. This signaling pathway is evolutionarily conserved and plays an essential role in the development of organs, tissues, and injury repair in multicellular organisms. Although this pathway plays a crucial role in the development of the kidney, it is silenced in the kidneys of normal adults and is reactivated against some kidney damage (Halt and Vainio, 2014). c-myc is a transcriptional target of β-catenin, which in turn regulates the proliferation and differentiation of renal tubular epithelium (Hu and Rosenblum, 2005). Recently the Pan group showed that myc cooperates with β-catenin to enhance the renewal of nephron progenitor cells (Pan et al., 2017), our LP animals showed lower c-myc expression, both genetic and protein so we can assume that these animals had a lower reserve of renewing cells, which are necessary for proliferation and survival, and may reflect on the smaller number of nephrons that are seen in the model.
β-catenin also has been shown to participate in a signaling pathway that controls organ size. This protein modulates the CM progenitor cell population and, together with a transcriptional complex, binds to the promoter region of the bcl2l1 anti-apoptotic gene in the developing kidney (Boivin et al., 2015). Besides, β-catenin interacts with the c-ret receptor, which, when stimulated by GDNF, releases it into the cytosol, and β-catenin migrates to the nucleus of UB cells (Sarin et al., 2014). In addition to its participation in the Wolffian duct elongation, β-catenin is essential for UB branching and differentiation (Bridgewater et al., 2008; Marose et al., 2009). However, have been shown that the increase in β-catenin expression has led to increased TGFB1 expression in epithelial cells inhibiting UB branching and causing premature differentiation of CM progenitor cells (Bridgewater et al., 2011)
Several studies have shown that increased β-catenin expression in CM disrupts UB ramifications and nephrogenesis by deregulating essential genes during renal development (Boivin et al., 2015). In the present study, the gene and protein expression of β-catenin is increased during the studied periods of renal development in LP animals. During renal development, β-catenin is expressed in both UB and CM modulating the gene programming that governs new budding and UB branching. In 17-DG LP is evident the increase of beta-catenin in the UB in the cytosol, whereas, in NP, the location is in the nucleus. Here, in CM, we also observed β-catenin labeling but not in NP age-matched offspring.
Although several authors have widely studied the process of normal nephrogenesis (Constantini and Kopan, 2010; Little and Mcmahon, 2012; Mugford et al., 2010), little is known about the mechanisms that determine the number of nephrons. Moreover, how the balance between nephron progenitor renewal and differentiation is essential for the proper development and function of the kidneys, this is a particularly important issue, because failure to achieve adequate numbers of nephrons is a risk factor for chronic renal disorder (Benz et al., 2011; Hoy et al., 2006; Luyckx and Brenner, 2005; Pan et al., 2017).