Oxidative Medicine and Cellular Longevity

Volume 2014 (2014), Article ID 283180, 21 pages

http://dx.doi.org/10.1155/2014/283180

Research Article

Melatonin Therapy Prevents Programmed Hypertension and Nitric Oxide Deficiency in Offspring Exposed to Maternal Caloric Restriction

You-Lin Tain,1,2 Li-Tung Huang,1,3 Chien-Ning Hsu,4,5 and Chien-Te Lee⁶

¹Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung 833, Taiwan

²Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung 833, Taiwan

³Department of Traditional Chinese Medicine, Chang Gung University, Linkou 333, Taiwan

⁴Department of Pharmacy, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung 833, Taiwan

⁵Graduate Institute of Clinical Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 833, Taiwan

⁶Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung 833, Taiwan

Received 6 January 2014; Revised 18 March 2014; Accepted 28 March 2014; Published 22 April 2014

Academic Editor: Jean-Claude Lavoie

Copyright © 2014 You-Lin Tain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Nitric oxide (NO) deficiency is involved in the development of hypertension, a condition that can originate early in life. We examined whether NO deficiency contributed to programmed hypertension in offspring from mothers with calorie-restricted diets and whether melatonin therapy prevented this process. We examined 3-month-old male rat offspring from four maternal groups: untreated controls, 50% calorie-restricted (CR) rats, controls treated with melatonin (0.01% in drinking water), and CR rats treated with melatonin (CR + M). The effect of melatonin on nephrogenesis was analyzed using next-generation sequencing. The CR group developed hypertension associated with elevated plasma asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), decreased L-arginine, decreased L-arginine-to-ADMA ratio (AAR), and decreased renal NO production. Maternal melatonin treatment prevented these effects. Melatonin prevented CR-induced renin and prorenin receptor expression. Renal angiotensin-converting enzyme 2 protein levels in the M and CR + M groups were also significantly increased by melatonin therapy. Maternal melatonin therapy had long-term epigenetic effects on global gene expression in the kidneys of offspring. Conclusively, we attributed these protective effects of melatonin on CR-induced programmed hypertension to the reduction of plasma ADMA, restoration of plasma AAR, increase of renal NO level, alteration of renin-angiotensin system, and epigenetic changes in numerous genes.

1. Introduction

Hypertension might originate during early life. Maternal malnutrition can impair development, resulting in intrauterine growth restriction (IUGR), permanent structural changes, and disrupted physiological function—a phenomenon called “developmental programming” [1]. In the kidneys of both humans and experimental models, developmental programming reduces nephron numbers, alters the renin-angiotensin system (RAS), and impairs natriuresis, leading to adult kidney disease and hypertension [2–5].

A number of hypotheses have been proposed to explain the developmental programming phenomenon [6]. Oxidative stress is proposed as the underlying link between developmental programing and elevated risks of hypertension and kidney disease in adulthood [7, 8]. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase (NOS), causes oxidative stress and is involved in the development of hypertension [9]. Our recent work demonstrated that an impaired ADMA-NO pathway and low nephron numbers are associated with programmed hypertension in the adult offspring of malnourished or diabetic mothers [10, 11]. Reduced nephron numbers impaired renal tubular sodium reabsorption, and the altered RAS components disrupted sodium retention, ultimately increasing blood pressure (BP) and inducing kidney damage. Histone deacetylases (HDACs) repress gene expression, a mechanism of epigenetic control that is involved in developmental programming. Class I HDACs are critical in nephrogenesis, particularly HDAC1-3 that are highly expressed in nephron precursors [12]. HDACs also play an important role in regulating RAS components during nephrogenesis [13]. These observations suggest that these mechanisms jointly lead to the development of hypertension and kidney disease.

Melatonin, an indoleamine produced from the pineal gland, is an antioxidant and free radical scavenger [14]. Experimental and human studies indicate that melatonin can regulate BP [10, 11]. We recently found that melatonin can prevent oxidative stress and hypertension concurrently in young spontaneously hypertensive rats (SHR) [15]. Emerging evidence supports novel roles of melatonin in epigenetic modulation through the regulation of HDACs [16, 17]. Thus, we examined whether melatonin prevented programmed hypertension in offspring exposed to maternal caloric restriction through reduction of oxidative stress, alteration of the RAS pathway, and modulation of HDACs. Moreover, we identified melatonin-induced gene changes during nephrogenesis and determined whether melatonin treatment induced global changes in biological processes by using next-generation sequencing.

2. Material and Methods

2.1. Animal Models

This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital. Virgin Sprague-Dawley (SD) rats (12–16 weeks old) were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan), and were housed and maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Male SD rats were caged with individual females until mating was confirmed. Calorie-restricted (CR) maternal rats received 11 g/d of a standard chow from day 11 of pregnancy until the day of delivery (day 23) and 20 g/d during the entire lactation period [10]. A subset of CR mothers was treated for the duration of the pregnancy with 0.01% melatonin dissolved in drinking water (CR + M, ). The control group mothers had free access to standard rat chow. As another control, maternal rats were allowed free access to standard rat chow and were treated with 0.01% melatonin in drinking water (M, ). After birth, each litter was left with the mother until weaning; pups were not weighed at birth to prevent maternal rejection. Male offspring, selected at random from each litter, were used in all subsequent experiments. In rats, nephrogenesis occurs predominantly from late gestation to 1-2 weeks postnatum and litters were typically weaned by postnatal week 3. Thus, melatonin was administered to mother rats for a total period of 6 weeks to cover the entire period of nephrogenesis. The dose of melatonin used was based on our previous study [15]. Water bottles were covered with aluminum foil to protect them from light. BP was measured in conscious rats by using an indirect tail-cuff method (BP-2000, Visitech Systems, Inc., Apex, NC, USA) after they had been systematically trained [10]. Three stable consecutive measures were taken and averaged. All offspring were sacrificed at 12 weeks of age and heparinized blood samples were collected. Kidneys were harvested after perfusion with PBS, divided into cortex and medulla regions, and snap-frozen. The activity of dimethylarginine dimethylaminohydrolase (DDAH), an ADMA-metabolizing enzyme, was measured using a colorimetric assay. The assay determined the rate of L-citrulline production and we performed the assay as previously described [18].

2.2. High-Performance Liquid Chromatography (HPLC)

Plasma and kidney L-arginine, L-citrulline, ADMA, and symmetric dimethylarginine (SDMA, a stereoisomer of ADMA) levels were measured using HPLC (HP series 1100, Agilent Technologies, Inc., Santa Clara, CA, USA) with the OPA-3MPA derivatization reagent as we described previously [10]. Standards contained L-arginine, L-citrulline, ADMA, and SDMA in the range of 1–100 μM, 1–100 μM, 0.5–5 μM, and 0.5–5 μM, respectively. The recovery rate was between 90 and 105%. The tissue concentration was factored for protein concentration, which was represented as μmol/mg protein. Plasma and urine creatinine (Cr) levels were analyzed by HPLC as described previously [10]. The creatinine clearance (CCr) was calculated by dividing the total amount of Cr excreted in urine by the Cr concentration in plasma. CCr values were normalized with respect to body weight.

2.3. Electron Paramagnetic Resonance (EPR)

Superoxide production was measured by EPR spectroscopy using a 1-hydroxy-3-carboxypyrrolidine (CPH) hydroxylamine spin probe, as we previously described [11]. The EPR spectra were recorded using an EMX Plus EPR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with an EMX-m40X microwave bridge operating at 9.87 GHz. NO was detected by EPR using N-methyl-D-glucamine dithiocarbamate (MGD) spin probe and FeSO₄, as previously described [11]. The EPR spectra were recorded using an EMX Plus EPR spectrometer (Bruker BioSpin) equipped with an EMX-m40X microwave bridge operating at 3.16 GHz.

2.4. Metanephros Organ Culture

Metanephros organ culture was performed as we described previously [11]. Briefly, SD female rats of known mating date were anesthetized and laparotomized. Fetuses were aseptically removed, and metanephroi from fetuses at embryonic day 14 (E14) were collected and freed of exogenous tissue. Explants were placed onto a Steritop filter unit (Millipore, Billerica, MA, USA) floating on a defined serum-free medium and incubated for 6 d in 35 mm Petri dishes at 37°C in a humidified incubator (5% CO₂). The defined medium was composed of Eagle’s Minimum Essential Medium containing 10% (v/v) fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. All of these reagents were obtained from Sigma (St. Louis, MO, USA). The culture medium was changed daily, and no antibiotic or fungicide was present throughout the experiment. Fresh aliquots of each culture medium additive were used for each metanephros culture. The medium was changed daily. Metanephroi were treated with melatonin (1 μM and 1 mM) and harvested after 6 d for real-time polymerase chain reaction.

2.5. Quantitative Real-Time Polymerase Chain Reaction (PCR)

RNA was extracted as described previously [10]. Two-step quantitative real-time PCR was conducted using the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) and the iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Nephron deficit was assessed by changes in the expression factors known to be involved in branching morphogenesis (BMP4, FGF2, and PAX2) and apoptosis (p53 and Bax). Several components of the RAS were analyzed including renin, prorenin receptor (PRR), angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin II type 1 (AT1R) and 2 receptor (AT2R), and angiotensin (1–7) MAS receptor. Class I HDACs, HDAC-1, -2, -3, and -8, were also examined. We used 18S rRNA (r18S) as a reference. Primers were designed using GeneTool Software (BioTools, Edmonton, Alberta, Canada) (Table 1). All samples were run in duplicate. To quantify the relative gene expression, the comparative threshold cycle method was employed. For each sample, the average CT value was subtracted from the corresponding average r18S value, calculating the . was calculated by subtracting the average control value from the average experimental . The fold-increase of the experimental sample relative to the control was calculated using the formula .

Table 1: PCR primers sequences.

Gene
Forward
Reverse

Bax
5 ttgctgatggcaacttcaactg 3
5 ctttagtgcacagggccttgag 3

P53
5 catgagcgttgctctgatg 3
5 cagatactcagcatacggatttcc 3

PAX2
5 gagactcccagagtggtgtg 3
5 cattcccctgttctgatttg 3

FGF2
5 ccagttggtatgtggcactg 3
5 cagggaagggtttgacaaga 3

BMP4
5 gacttcgaggcgacacttctg 3
5 agccggtaaagatccctcatg 3

Renin
5 aacattaccagggcaactttcact 3
5 acccccttcatggtgatctg 3

Prorenin receptor
5 gaggcagtgaccctcaacat 3
5 ccctcctcacacaacaaggt 3

Angiotensinogen
5 gcccaggtcgcgatgat 3
5 tgtacaagatgctgagtgaggcaa 3

ACE
5 caccggcaaggtctgctt 3
5 cttggcatagtttcgtgaggaa 3

ACE2
5 acccttcttacatcagccctactg 3
5 tgtccaaaacctaccccacatat 3

AT1R
5 gctgggcaacgagtttgtct 3
5 cagtccttcagctggatcttca 3

AT2R
5 caatctggctgtggctgactt 3
5 tgcacatcacaggtccaaaga 3

MAS
5 catctctcctctcggctttgtg 3
5 cctcatccggaagcaaagg 3

HDAC-1
5 gaactggggacctacggg 3
5 gctcttgacaaattccacacac 3

HDAC-2
5 agttgcccttgattgtgaga 3
5 ccactgttgtccttggatttat 3

HDAC-3
5 tgatgaccagagttacaagcac 3
5 gggcaacatttc ggacag 3

HDAC-8
5 gctacccccggtttatatttacag 3
5 ttcgatcagagagtgaaccatactg 3

R18S
5 gccgcggtaattccagctcca 3
5 cccgcccgctcccaagatc 3

2.6. Western Blot

Western blot analysis was performed as previously described [10]. We used the following antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA): rabbit polyclonal anti-rat PRR (1 : 500, overnight incubation), rabbit anti-rat ACE2 (1 : 1000, overnight incubation), rabbit anti-rat AT1R (1 : 250, overnight incubation), rabbit anti-rat AT2R (1 : 250, overnight incubation), and rabbit anti-rat MAS (1 : 1000, overnight incubation; Santa Cruz Biotechnology). The bands of interest were visualized using enhanced chemiluminescence reagent (PerkinElmer, Waltham, MA, USA) and quantified by densitometry (Quantity One Analysis software, Bio-Rad). Band density was calculated as the integrated optical density (IOD) minus the background value. The density of Ponceau red staining (PonS) was used to correct for variations in total protein loading. Protein abundance was calculated as IOD/PonS.

2.7. Next-Generation Sequencing and Analysis

In rats, nephrogenesis occurs predominantly from late gestation to 7–10 days postnatum. Thus, offspring from the control and M groups were sacrificed at 1 week of age. Kidneys were isolated and snap-frozen for whole-genome RNA next-generation sequencing (RNA-seq), performed by Welgene Biotech Co., Ltd. (Taipei, Taiwan). Purified RNA was quantified at 260 nm (OD₆₀₀) by using ND-1000 spectrophotometer (Nanodrop Technology, Wilmington, DE, USA) and analyzed using a Bioanalyzer 2100 (Agilent Technology) with RNA 6000 LabChip kit (Agilent Technologies). All procedures were performed according to the Illumina protocol. For all samples, library construction was performed using the TruSeq RNA Sample Prep Kit v2 for 160 bp (single-end) sequencing and the Solexa platform. The sequence was directly determined by sequencing-by-synthesis technology using the TruSeq SBS Kit. Raw sequences were obtained using the Illumina GA Pipeline software CASAVA v1.8, which was expected to generate 10 million reads per sample. Quantification for gene expression was calculated as reads per kilobase of exon per million mapped reads [19]. For differential expression analysis, Cufflink v 2.1.1 and CummeRbund v 2.0.0 were used to perform statistical analyses of the gene expression profiles. The reference genome and gene annotations were retrieved from the Ensembl database (http://asia.ensembl.org/index.html). Gene ontology analysis for significant genes was performed using KEGG (http://www.genome.jp/kegg/) and NIH DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/) to identify regulated biological themes.

2.8. Statistical Analysis

TheShapiro-Wilk normality test was used to determine which data were normally distributed. Normally distributed data are given as mean ± standard error of the mean. For most parameters, statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc test for multiple comparisons. BP was analyzed by two-way repeated-measures ANOVA and Tukey’s post hoc test. A value 0.05 was considered statistically significant. Analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (Chicago, IL, USA).

3. Results

3.1. The Effects of Melatonin on Morphological and Biochemical Values in CR Rats

Litter sizes were not significantly altered by caloric restriction in the mother rat or by melatonin treatment. The amounts of water intake and urine output were not significantly different in the control and CR groups. Male pup mortality rates did not differ between the four groups analyzed. As shown in Table 2, the CR and M groups had lower and higher body weight (BW) than the control at 12 weeks of age, respectively, whereas the CR + M group had an intermediate BW. Kidney weight and kidney weight-to-BW ratio did not differ between the control and CR groups. Melatonin significantly increased kidney weight and kidney weight-to-BW ratio in the M and CR + M groups. Although heart weight was not different between control and CR groups, the heart weight-to-BW ratio was greater in the CR group. Melatonin caused increased heart weight and heart weight-to-BW ratio in the M group, but not in the CR + M group. CR increased systolic and diastolic BP and mean arterial pressure at 12 weeks of age. Melatonin therapy prevented these effects of CR. In addition, melatonin therapy reduced diastolic BP and mean arterial pressure in the M group compared to the control. As shown in Figure 1, mean arterial pressure was similar in the four groups at 4 weeks of age. By 8 weeks of age, mean arterial pressure had increased in the CR group relative to controls. A significant reduction in mean arterial pressure was measured in the M and CR + M groups versus the control at 8 and 12 weeks of age. In contrast, plasma creatinine level did not differ between the four groups. These data demonstrated that CR induced programmed hypertension but had no effect on renal function on 12-week-old offspring.

Table 2: Morphological and biochemical values in different experimental groups.

Control
CR
M
CR + M

Mortality
10%
0%
0%
0%

Body weight (BW) (g)
435 ± 14
356 ± 4*

Left kidney weight (g)
1.22 ± 0.06
1.01 ± 0.02
1.97 ± 0.05*#
1.48 ± 0.03#$

Left kidney weight/100g BW
0.28 ± 0.01
0.28 ± 0.01
0.4 ± 0.01*#
0.4 ± 0.01*#

Heart weight (g)
1.23 ± 0.05
1.24 ± 0.02
1.63 ± 0.01*#
1.16 ± 0.05$

Heart weight/100 g BW
0.28 ± 0.01
0.35 ± 0.01*
0.35 ± 0.01*
0.31 ± 0.01

Systolic blood pressure (mmHg)
162 ± 2
180 ± 2*
155 ± 1#
166 ± 1$

Diastolic blood pressure (mmHg)
122 ± 2
134 ± 3*
108 ± 2*#
113 ± 1*#

Mean arterial pressure (mmHg)
135 ± 2
149 ± 2*
124 ± 1*#
131 ± 1#$

CCr, mL·min⁻¹·kg body weight⁻¹
9.12 ± 3.45
8.5 ± 3.0
7.34 ± 2.32
7.81 ± 2.76

CCr: clearance of creatinine; versus control; versus CR; $ versus M.

Figure 1: Effect of melatonin and caloric restriction (CR) on mean arterial pressure in male offspring at 12 weeks of age.   versus control; # versus CR.

3.2. The Effects of Melatonin on L-Arginine, L-Citrulline, and Dimethylarginine Levels

As shown in Table 3, plasma levels of ADMA and SDMA were elevated nearly 70% and 150% following maternal CR, respectively. In contrast, the L-arginine levels and L-arginine-to-ADMA ratio were decreased by 30% and 55%, respectively. Melatonin treatment significantly increased L-arginine levels and L-arginine-to-ADMA ratio, but decreased ADMA and SDMA levels in the CR + M group. In the kidney, levels of L-citrulline, L-arginine, ADMA, and SDMA did not differ between the four groups. However, renal L-arginine-to-ADMA ratio was higher in the CR + M group versus the M group. We next analyzed superoxide and NO production in the kidney by using EPR. We found no difference in renal superoxide level among the four groups (control: , CR: , M: , CR + M: arbitrary units; ). CR significantly reduced renal NO levels, but not in the presence of melatonin (control: , CR: , M: , CR + M: arbitrary units; control versus CR, ; CR versus CR + M, ).

Table 3: L-Citrulline, L-arginine, and dimethylarginine levels in the plasma and kidney.

Control
CR
M
CR + M

Plasma (μmol)

    L-Citrulline
50 ± 4.1
61 ± 3.6
59.3 ± 5.1
55.8 ± 6.9

    L-Arginine
121.1 ± 14
84.4 ± 2.4*
113.6 ± 8.7#
112.8 ± 13.6#

    ADMA
1.31 ± 0.1
2.21 ± 0.18*
1.18 ± 0.06#
1.08 ± 0.12#

    SDMA
0.66 ± 0.04
1.62 ± 0.27*
0.97 ± 0.09#
0.92 ± 0.08#

    L-Arginine-to-ADMA ratio
92 ± 8
40 ± 4*
98 ± 10#
105 ± 6#

Kidney (μmol/mg protein)

    L-Citrulline
52.5 ± 8.6
53.1 ± 4.6
97.6 ± 8.4
68.8 ± 12.4

    L-Arginine
425 ± 62.3
552.9 ± 58.9
522.8 ± 61.6
488.1 ± 56

    ADMA
5.09 ± 0.88
6.33 ± 0.71
6.72 ± 1.03
4.84 ± 0.61

    SDMA
4.3 ± 0.65
5.3 ± 0.51
5.57 ± 0.79
4.59 ± 0.73

    L-Arginine-to-ADMA ratio
86 ± 4
89 ± 5
80 ± 4
103 ± 8$

versus control; versus CR; $ versus M.

3.3. The Effects of Melatonin on the ADMA Pathway

Next, we examined the expression/activity of proteins involved in the ADMA pathway. We found that renal level of protein arginine N-methyltransferase 1 (PRMT-1), an ADMA-synthesizing enzyme, was significantly lower in the M and CR + M groups than that in control and CR groups (Figure 2(b)). However, protein levels of DDAH-1 and -2, ADMA-metabolizing enzymes, in the kidney were not different between the four groups (Figures 2(c) and 2(d)). We found that renal DDAH activity did not differ between control and CR groups (Figure 2(e)). However, melatonin increased renal DDAH activity in both the M and CR + M groups. Thus, we speculate that the increase of systemic ADMA observed with CR is due to excessive synthesis or decreased metabolism in extrarenal tissues. On the other hand, the reduced plasma ADMA levels in response to melatonin might be due to decreased ADMA synthesis and increased ADMA breakdown in the kidney.

(a)

(b)

(c)

(d)

(e)

Figure 2: Representative western blots (a) showing protein arginine methyltransferase 1 (PRMT-1; 42 kDa), dimethylarginine dimethylaminohydrolase 1 (DDAH-1; 34 kDa), and DDAH-2 (30 kDa) bands in CR offspring at 12 weeks of age. Relative abundance of renal cortical (b) PRMT-1, (c) DDAH-1, and (d) DDAH-2. (e) Effect of melatonin and CR on renal DDAH activity in male offspring at 12 weeks of age. versus control; # versus CR.

3.4. The Effects of Melatonin on Nephrogenesis

We investigated whether changes in nephrogenesis- or apoptosis-related gene expression were associated with CR-induced reduced nephron numbers, as we found previously [10]. Consistent with our previous report [10], renal expression of p53 and the proapoptotic factor Bax did not differ between the control and CR groups (Figure 3(a)). Similarly, growth factors BMP4 and FGF2 were unaltered by CR or melatonin in the kidney. However, melatonin significantly increased the expression of the transcriptional activator PAX2 in CR + M group compared to controls (Figure 3(a)).

(a)

(b)

(c)

(d)

Figure 3: Effect of melatonin and CR on the expression of (a) apoptosis- and nephrogenesis-related genes, (b) sodium transporters, (c) renin-angiotensin system (RAS) components, and (d) class I histone deacetylase (HDAC) in the kidney at 12 weeks of age. versus control; versus CR; versus M.

3.5. The Effects of Melatonin on Sodium Transporters, RAS, and HDACs

Next, we evaluated two critical pathways involved in hypertension, sodium transporters and RAS components. We found that CR upregulated sodium-hydrogen exchanger 3 (NHE3) expression in the kidney (Figure 3(b)). The increase in renal NHE3 expression was not prevented by melatonin therapy. CR had no effect on the expression of RAS genes in the kidney, including renin, PRR, AGT, ACE, ACE2, AT1R, AT2R, and MAS (Figure 3(c)). Melatonin treatment, on the other hand, upregulated renal expression of renin, PRR, and ACE2 in the CR + M group compared to the control. Because melatonin therapy prevented the elevation of BP in offspring exposed to maternal CR, our data suggested that the antihypertensive effect of melatonin was related to renin, PRR, and ACE2 expression in the CR model. We found that CR did not alter renal expression of class I HDACs in the CR versus control group (Figure 3(d)). However, melatonin therapy increased HDAC-2, -3, and -8 expression in the kidney.

We analyzed the renal protein levels of PRR, ACE2, AT1R, AT2R, and MAS. Melatonin therapy significantly increased renal PRR and ACE2 protein levels in the M and CR + M group compared with the control and CR groups (Figures 4(b) and 4(c)). We observed that renal AT1R, AT2R, and MAS protein levels did not differ among the four groups (Figures 4(d)–4(f)).

(a)

(b)

(c)

(d)

(e)

(f)

Figure 4: Representative western blots (a) showing prorenin receptor (PRR; 39 kDa), angiotensin-converting enzyme (ACE2; 50 kDa), angiotensin II type 1 (AT1R; 43 kDa) and type 2 (AT2R; 90 kDa), and MAS (37 kDa) proteins in male offspring kidneys at 12 weeks of age. Relative abundance of renal (b) PRR, (c) ACE2, (d) AT1R, (e) AT2R, and (f) MAS is quantified. versus control; # versus CR.

We also determined whether melatonin regulated nephrogenesis-related genes, RAS components, sodium transporters, and HDACs during nephrogenesis. The mRNA levels in rat metanephroi grown in different concentrations of melatonin are shown in Figure 5. We found that low doses of melatonin had no effect on the expression of these genes, whereas high-dose melatonin treatment significantly increased expression of PAX2, renin, PRR, Mas, NHE3, and Na-K-Cl cotransporter 2 in metanephroi.

(a)

(b)

(c)

Figure 5: Gene expression of (a) apoptosis- and nephrogenesis-related genes, (b) RAS components, and (c) sodium transporters in the metanephroi of offspring from mothers treated with melatonin (1 μM or 1 mM). versus control ().

3.6. The Effects of Melatonin on Gene Expression during Nephrogenesis

We demonstrated that numerous individual genes were significantly regulated in the kidneys of offspring from melatonin-treated mothers during a critical period of renal development. As shown in Table 4, 439 and 15 genes were upregulated and downregulated, respectively. The most significantly regulated biological theme in the KEGG gene ontology analysis was tryptophan metabolism (Figure 6).

Table 4: Genes that changed by RPKM > 0.3 in the kidney of melatonin treated offspring versus control at 1 week of age.

Gene_ID
Gene symbol
Fold changes
Log₂
value

Upregulated: 439 genes

ENSRNOG00000038989
D3ZSD6_RAT
28.686
4.842
0.0083

ENSRNOG00000006367
Slc5a8
19.264
4.268
0.0001

ENSRNOG00000003038
Sft2d2
17.101
4.096
0.0020

ENSRNOG00000007720
F1LX97_RAT
13.841
3.791
0.0027

ENSRNOG00000019014
Ndst1
12.657
3.662
0.0003

ENSRNOG00000017434
Mgat3
12.364
3.628
0.0005

ENSRNOG00000001656
Kcnj15
11.724
3.551
0.0028

ENSRNOG00000021292
—
11.449
3.517
0.0015

ENSRNOG00000017078
Sepn1
11.107
3.473
0.0011

ENSRNOG00000030121
Enpep
10.970
3.456
0.0029

ENSRNOG00000005854
Angpt1
10.920
3.449
0.0165

ENSRNOG00000009944
LOC314407
10.858
3.441
0.0017

ENSRNOG00000013279
Scd
10.775
3.430
0.0012

ENSRNOG00000001724
LOC678704
10.690
3.418
0.0011

ENSRNOG00000002463
LOC682752
10.633
3.411
0.0033

ENSRNOG00000011630
Ak3l1
10.304
3.365
0.0441

ENSRNOG00000005447
RGD1311564
10.117
3.339
0.0026

ENSRNOG00000009019
Slc6a6
10.090
3.335
0.0016

ENSRNOG00000002969
Itpkb
9.892
3.306
0.0020

ENSRNOG00000037307
Spata22
9.876
3.304
0.0036

ENSRNOG00000039717
Ipo11
9.584
3.261
0.0069

ENSRNOG00000025372
Glce
9.536
3.253
0.0023

ENSRNOG00000037884
Oxgr1
9.510
3.249
0.0169

ENSRNOG00000021203
Atl3
9.487
3.246
0.0056

ENSRNOG00000006787
Dhcr24
9.328
3.222
0.0023

ENSRNOG00000015038
Adam10
9.279
3.214
0.0005

ENSRNOG00000002519
Magt1
9.253
3.210
0.0010

ENSRNOG00000038933
D3ZF12_RAT
9.225
3.206
0.0023

ENSRNOG00000024757
RGD1310444
9.119
3.189
0.0066

ENSRNOG00000030285
Epha3
9.064
3.180
0.0032

ENSRNOG00000018338
Vwa1
9.017
3.173
0.0355

ENSRNOG00000022802
Tmem184b
8.982
3.167
0.0070

ENSRNOG00000013265
Tgfbr2
8.947
3.161
0.0014

ENSRNOG00000026941
Tril
8.934
3.159
0.0024

ENSRNOG00000020532
Kcnq1
8.904
3.154
0.0441

ENSRNOG00000018503
LOC293190
8.862
3.148
0.0172

ENSRNOG00000002198
LOC685352
8.711
3.123
0.0037

ENSRNOG00000017172
Fam125b
8.706
3.122
0.0291

ENSRNOG00000018554
—
8.663
3.115
0.0067

ENSRNOG00000013963
IL6RB_RAT
8.571
3.099
0.0023

ENSRNOG00000042565
—
8.547
3.095
0.0114

ENSRNOG00000032834
Hspa13
8.544
3.095
0.0011

ENSRNOG00000002355
Slc47a1
8.474
3.083
0.0025

ENSRNOG00000011927
SDC3_RAT
8.460
3.081
0.0065

ENSRNOG00000042540
Mef2a
8.454
3.080
0.0368

ENSRNOG00000029216
Dgcr2
8.331
3.059
0.0175

ENSRNOG00000023725
LOC689756
8.215
3.038
0.0191

ENSRNOG00000028129
Fktn
8.207
3.037
0.0063

ENSRNOG00000000547
Tspyl4
8.202
3.036
0.0103

ENSRNOG00000011859
Eif5a2
8.192
3.034
0.0403

ENSRNOG00000028387
E9PTK5_RAT
8.168
3.030
0.0197

ENSRNOG00000015986
Rassf8
8.134
3.024
0.0094

ENSRNOG00000029409
Gstm6l
8.062
3.011
0.0292

ENSRNOG00000008895
Hnf4a
7.993
2.999
0.0398

ENSRNOG00000038149
Defb9
7.948
2.991
0.0437

ENSRNOG00000040287
Cyp1b1
7.890
2.980
0.0439

ENSRNOG00000010468
Elovl6
7.871
2.977
0.0394

ENSRNOG00000014524
F1M9D3_RAT
7.828
2.969
0.0077

ENSRNOG00000014209
Utp6
7.793
2.962
0.0050

ENSRNOG00000013419
Agphd1
7.789
2.961
0.0031

ENSRNOG00000020653
S1pr2
7.775
2.959
0.0313

ENSRNOG00000018714
Arl5b
7.770
2.958
0.0078

ENSRNOG00000002408
Rbm47
7.719
2.948
0.0057

ENSRNOG00000008971
Hnf4g
7.715
2.948
0.0085

ENSRNOG00000011271
Mcc
7.688
2.943
0.0120

ENSRNOG00000002276
LOC100359714
7.641
2.934
0.0073

ENSRNOG00000009446
Rxra
7.607
2.927
0.0066

ENSRNOG00000019400
Dag1
7.591
2.924
0.0010

ENSRNOG00000013098
F1M9J1_RAT
7.581
2.922
0.0320

ENSRNOG00000014511
Alg10
7.551
2.917
0.0076

ENSRNOG00000012490
Amph
7.533
2.913
0.0461

ENSRNOG00000014934
Fam63b
7.481
2.903
0.0193

ENSRNOG00000039630
LOC290577
7.414
2.890
0.0045

ENSRNOG00000032707
Egf
7.368
2.881
0.0017

ENSRNOG00000015605
Ptprk
7.357
2.879
0.0298

ENSRNOG00000000168
Gatm
7.311
2.870
0.0017

ENSRNOG00000027097
F1M683_RAT
7.273
2.863
0.0087

ENSRNOG00000018109
Clic4
7.251
2.858
0.0048

ENSRNOG00000008629
Secisbp2l
7.236
2.855
0.0042

ENSRNOG00000019799
Pcdhgc3
7.231
2.854
0.0246

ENSRNOG00000024089
Fndc3b
7.221
2.852
0.0065

ENSRNOG00000015852
D4AD82_RAT
7.192
2.846
0.0020

ENSRNOG00000006967
Xiap
7.151
2.838
0.0136

ENSRNOG00000031487
F1LM52_RAT
7.129
2.834
0.0477

ENSRNOG00000014866
Pign
7.077
2.823
0.0190

ENSRNOG00000033206
Entpd5
7.060
2.820
0.0070

ENSRNOG00000037753
Slc10a2
7.002
2.808
0.0089

ENSRNOG00000040195
F1LZT0_RAT
7.001
2.808
0.0018

ENSRNOG00000042817
D4A5M8_RAT
6.925
2.792
0.0104

ENSRNOG00000005070
Spopl
6.920
2.791
0.0139

ENSRNOG00000006459
D4AEA4_RAT
6.871
2.781
0.0251

ENSRNOG00000012784
Gtf3c4
6.850
2.776
0.0096

ENSRNOG00000016968
Gramd4
6.838
2.774
0.0216

ENSRNOG00000004448
RGD1307051
6.819
2.770
0.0050

ENSRNOG00000021809
Gpx3
6.801
2.766
0.0008

ENSRNOG00000014183
Gnaq
6.801
2.766
0.0084

ENSRNOG00000012991
LOC100363275
6.798
2.765
0.0046

ENSRNOG00000013443
Tm9sf3
6.791
2.764
0.0040

ENSRNOG00000042673
LOC100359544
6.789
2.763
0.0012

ENSRNOG00000003873
Cpd
6.767
2.758
0.0028

ENSRNOG00000007990
Adipor2
6.762
2.758
0.0026

ENSRNOG00000007804
C1galt1
6.762
2.757
0.0109

ENSRNOG00000043256
D3ZNR8_RAT
6.720
2.749
0.0145

ENSRNOG00000015124
Gpam
6.720
2.748
0.0109

ENSRNOG00000004888
Spred2
6.690
2.742
0.0454

ENSRNOG00000003960
Tmem27
6.682
2.740
0.0026

ENSRNOG00000015750
Wnt7b
6.654
2.734
0.0218

ENSRNOG00000030763
Dpp4
6.601
2.723
0.0011

ENSRNOG00000039504
Q5M885_RAT
6.562
2.714
0.0116

ENSRNOG00000032768
D3Z9G8_RAT
6.497
2.700
0.0214

ENSRNOG00000039771
LOC100361629
6.494
2.699
0.0140

ENSRNOG00000009274
Fut11
6.475
2.695
0.0354

ENSRNOG00000027938
RGD1562037
6.420
2.683
0.0117

ENSRNOG00000001335
Zkscan1
6.419
2.682
0.0077

ENSRNOG00000004978
Prkacb
6.379
2.673
0.0216

ENSRNOG00000005446
Gna11
6.363
2.670
0.0172

ENSRNOG00000003884
Acmsd
6.362
2.669
0.0262

ENSRNOG00000028190
D4ACF8_RAT
6.354
2.668
0.0432

ENSRNOG00000006338
Lrp6
6.351
2.667
0.0041

ENSRNOG00000009523
Rab11fip2
6.345
2.666
0.0470

ENSRNOG00000003759
Galc
6.345
2.666
0.0140

ENSRNOG00000010620
NDC1_RAT
6.319
2.660
0.0275

ENSRNOG00000001821
Adipoq
6.306
2.657
0.0244

ENSRNOG00000038572
RGD1562646
6.293
2.654
0.0106

ENSRNOG00000026120
Fam8a1
6.282
2.651
0.0129

ENSRNOG00000025476
RGD1359349
6.243
2.642
0.0126

ENSRNOG00000019508
Wars2
6.216
2.636
0.0317

ENSRNOG00000008271
Fam91a1
6.216
2.636
0.0031

ENSRNOG00000017120
Abhd2
6.208
2.634
0.0278

ENSRNOG00000010843
Nhlrc3
6.203
2.633
0.0255

ENSRNOG00000030704
F1LV74_RAT
6.139
2.618
0.0369

ENSRNOG00000002509
Gnl3l
6.129
2.616
0.0124

ENSRNOG00000010841
Col8a2
6.089
2.606
0.0457

ENSRNOG00000002728
Btc
6.088
2.606
0.0348

ENSRNOG00000027320
Eif2c1
6.082
2.605
0.0243

ENSRNOG00000009453
Mobkl2b
6.072
2.602
0.0271

ENSRNOG00000007797
Rbpsuh
6.069
2.602
0.0133

ENSRNOG00000017286
HYES_RAT
6.064
2.600
0.0032

ENSRNOG00000002461
Nid1
6.057
2.599
0.0014

ENSRNOG00000006649
Thrb
6.048
2.596
0.0180

ENSRNOG00000025042
Pde3a
6.048
2.596
0.0189

ENSRNOG00000015916
Ttc38
6.048
2.596
0.0384

ENSRNOG00000013581
Extl3
6.038
2.594
0.0093

ENSRNOG00000002332
MSPD1_RAT
6.034
2.593
0.0213

ENSRNOG00000032757
D3Z903_RAT
6.032
2.593
0.0431

ENSRNOG00000029651
Rdh2
6.025
2.591
0.0258

ENSRNOG00000018588
Sox4
6.019
2.590
0.0342

ENSRNOG00000012428
Maf
6.005
2.586
0.0483

ENSRNOG00000009506
Mre11a
6.005
2.586
0.0332

ENSRNOG00000028330
—
5.987
2.582
0.0375

ENSRNOG00000034025
D4A4T5_RAT
5.979
2.580
0.0227

ENSRNOG00000007079
Met
5.979
2.580
0.0117

ENSRNOG00000008088
Btbd3
5.979
2.580
0.0181

ENSRNOG00000017546
Mylk3
5.945
2.572
0.0224

ENSRNOG00000042333
Dnal1
5.895
2.559
0.0187

ENSRNOG00000001092
Kl
5.873
2.554
0.0106

ENSRNOG00000016498
—
5.836
2.545
0.0049

ENSRNOG00000037765
Lims1
5.833
2.544
0.0367

ENSRNOG00000010267
Klhdc10
5.827
2.543
0.0346

ENSRNOG00000043277
D3ZIC7_RAT
5.809
2.538
0.0206

ENSRNOG00000024799
D3ZNV9_RAT
5.803
2.537
0.0032

ENSRNOG00000004919
Gns
5.795
2.535
0.0282

ENSRNOG00000015080
Wdfy1
5.766
2.528
0.0292

ENSRNOG00000009565
Pdk4
5.764
2.527
0.0206

ENSRNOG00000013082
LCAP_RAT
5.754
2.525
0.0322

ENSRNOG00000026501
Slc6a19
5.742
2.522
0.0406

ENSRNOG00000009597
Cyp4a1
5.740
2.521
0.0123

ENSRNOG00000011560
Mtmr9
5.738
2.521
0.0368

ENSRNOG00000022710
Prrg4
5.736
2.520
0.0269

ENSRNOG00000013469
LOC100362805
5.715
2.515
0.0059

ENSRNOG00000024640
RGD1304731
5.698
2.510
0.0080

ENSRNOG00000018952
Sema3g
5.692
2.509
0.0143

ENSRNOG00000020011
Q66HF5_RAT
5.677
2.505
0.0381

ENSRNOG00000012826
Creb3l2
5.665
2.502
0.0189

ENSRNOG00000032492
Usp22
5.657
2.500
0.0107

ENSRNOG00000021840
LOC500046
5.644
2.497
0.0118

ENSRNOG00000034026
Lclat1
5.642
2.496
0.0223

ENSRNOG00000009153
Cidec
5.642
2.496
0.0432

ENSRNOG00000028899
Zbtb33
5.633
2.494
0.0168

ENSRNOG00000001766
Tfrc
5.613
2.489
0.0102

ENSRNOG00000017901
Acy3
5.613
2.489
0.0044

ENSRNOG00000012095
Pkia
5.596
2.484
0.0339

ENSRNOG00000001796
Dgkg
5.573
2.479
0.0471

ENSRNOG00000004958
RGD1304605
5.563
2.476
0.0100

ENSRNOG00000025587
Plagl1
5.550
2.472
0.0289

ENSRNOG00000027540
Fam102b
5.536
2.469
0.0410

ENSRNOG00000001518
Itga6
5.519
2.465
0.0452

ENSRNOG00000032723
Eftud1
5.515
2.463
0.0336

ENSRNOG00000002053
F1M3H3_RAT
5.491
2.457
0.0081

ENSRNOG00000003472
Atp11c-ps1
5.473
2.452
0.0317

ENSRNOG00000003984
Apln
5.448
2.446
0.0337

ENSRNOG00000012453
RGD1564560
5.438
2.443
0.0046

ENSRNOG00000017846
Slc44a1
5.422
2.439
0.0293

ENSRNOG00000016921
Klhl11
5.418
2.438
0.0275

ENSRNOG00000026415
D4A301_RAT
5.403
2.434
0.0280

ENSRNOG00000013798
Fnbp1l
5.391
2.431
0.0098

ENSRNOG00000003620
Fmo3
5.384
2.429
0.0050

ENSRNOG00000018220
Pde4dip
5.377
2.427
0.0462

ENSRNOG00000000145
Pik3r3
5.352
2.420
0.0210

ENSRNOG00000008834
LOC306096
5.351
2.420
0.0356

ENSRNOG00000025882
Nipal1
5.345
2.418
0.0306

ENSRNOG00000010996
Mobkl1a
5.341
2.417
0.0147

ENSRNOG00000001582
Bach1
5.339
2.417
0.0199

ENSRNOG00000022309
D3ZRU8_RAT
5.313
2.410
0.0048

ENSRNOG00000015741
Slc2a13
5.298
2.406
0.0371

ENSRNOG00000014303
F1M753_RAT
5.294
2.404
0.0391

ENSRNOG00000036798
Dusp3
5.284
2.402
0.0199

ENSRNOG00000012142
Glyat
5.283
2.401
0.0081

ENSRNOG00000024426
D3ZXW1_RAT
5.259
2.395
0.0477

ENSRNOG00000006628
Dusp16
5.256
2.394
0.0271

ENSRNOG00000026143
Ckap2l
5.230
2.387
0.0271

ENSRNOG00000018867
Klhdc7a
5.223
2.385
0.0489

ENSRNOG00000025296
Lrrc8a
5.203
2.379
0.0176

ENSRNOG00000014508
Mgll
5.203
2.379
0.0137

ENSRNOG00000000589
RGD1310495
5.199
2.378
0.0372

ENSRNOG00000014234
Hif1an
5.192
2.376
0.0394

ENSRNOG00000008450
LOC100359539
5.178
2.372
0.0409

ENSRNOG00000010744
Nrp1
5.177
2.372
0.0072

ENSRNOG00000039837
RGD1563945
5.161
2.368
0.0466

ENSRNOG00000013177
Map3k1
5.154
2.366
0.0114

ENSRNOG00000021719
F1LX81_RAT
5.153
2.365
0.0133

ENSRNOG00000024629
Hadha
5.116
2.355
0.0126

ENSRNOG00000014907
Aldh8a1
5.105
2.352
0.0055

ENSRNOG00000036673
Sectm1b
5.098
2.350
0.0121

ENSRNOG00000024794
Senp5
5.096
2.349
0.0264

ENSRNOG00000005131
Lin7c
5.086
2.347
0.0289

ENSRNOG00000002225
Scarb2
5.081
2.345
0.0116

ENSRNOG00000020284
Prkar2a
5.077
2.344
0.0215

ENSRNOG00000014648
Efnb2
5.072
2.343
0.0303

ENSRNOG00000002488
Galnt10
5.063
2.340
0.0437

ENSRNOG00000017406
Atrnl1
5.056
2.338
0.0269

ENSRNOG00000010813
Tspan14
5.048
2.336
0.0304

ENSRNOG00000000645
Reep3
5.047
2.336
0.0262

ENSRNOG00000018873
Fam168a
5.036
2.332
0.0160

ENSRNOG00000020253
RAB1B_RAT
5.030
2.331
0.0128

ENSRNOG00000001235
Gna12
5.012
2.325
0.0149

ENSRNOG00000040215
F1LZL1_RAT
5.011
2.325
0.0302

ENSRNOG00000011619
Myo9a
4.988
2.319
0.0163

ENSRNOG00000039976
D3ZHG3_RAT
4.983
2.317
0.0137

ENSRNOG00000016011
Plekhg1
4.971
2.314
0.0315

ENSRNOG00000037909
Ppm1f
4.964
2.312
0.0269

ENSRNOG00000016419
Pdlim5
4.962
2.311
0.0248

ENSRNOG00000023280
Als2
4.952
2.308
0.0166

ENSRNOG00000005417
Zhx2
4.948
2.307
0.0430

ENSRNOG00000017671
Rasa3
4.944
2.306
0.0403

ENSRNOG00000016848
Fzd4
4.942
2.305
0.0255

ENSRNOG00000003508
LOC100364400
4.942
2.305
0.0244

ENSRNOG00000012394
Bcl2l13
4.931
2.302
0.0466

ENSRNOG00000018400
D4AEL2_RAT
4.931
2.302
0.0303

ENSRNOG00000013707
Spata13
4.930
2.302
0.0445

ENSRNOG00000002039
LOC100360066
4.930
2.301
0.0436

ENSRNOG00000004563
Sec24a
4.917
2.298
0.0191

ENSRNOG00000020386
D3ZKH4_RAT
4.906
2.295
0.0098

ENSRNOG00000007419
Pank3
4.900
2.293
0.0128

ENSRNOG00000024533
Aer61
4.889
2.290
0.0382

ENSRNOG00000027151
Lrrc58
4.886
2.289
0.0393

ENSRNOG00000030124
Ptpn11
4.869
2.284
0.0160

ENSRNOG00000006131
Mettl2
4.846
2.277
0.0271

ENSRNOG00000000407
Dcbld1
4.834
2.273
0.0412

ENSRNOG00000008061
Nuak1
4.826
2.271
0.0360

ENSRNOG00000037514
Qser1
4.821
2.269
0.0136

ENSRNOG00000004959
Actr2
4.807
2.265
0.0327

ENSRNOG00000028582
F1M163_RAT
4.795
2.261
0.0045

ENSRNOG00000043037
LOC100366023
4.788
2.259
0.0349

ENSRNOG00000012135
F1M2H7_RAT
4.763
2.252
0.0406

ENSRNOG00000031069
D4A9A7_RAT
4.749
2.247
0.0462

ENSRNOG00000023109
F1LVL2_RAT
4.736
2.244
0.0482

ENSRNOG00000004442
RGD1311756
4.729
2.241
0.0456

ENSRNOG00000021318
Epas1
4.723
2.240
0.0138

ENSRNOG00000018099
Itch
4.702
2.233
0.0383

ENSRNOG00000038892
LOC686123
4.691
2.230
0.0268

ENSRNOG00000000296
Aqp6
4.685
2.228
0.0310

ENSRNOG00000014901
Uggt1
4.684
2.228
0.0168

ENSRNOG00000019659
Aspa
4.680
2.227
0.0055

ENSRNOG00000010450
D4ADY9_RAT
4.662
2.221
0.0220

ENSRNOG00000011066
6-Mar
4.658
2.220
0.0264

ENSRNOG00000013121
Mier3
4.647
2.216
0.0408

ENSRNOG00000030894
Slco1a6
4.640
2.214
0.0068

ENSRNOG00000004964
Erbb3
4.609
2.205
0.0351

ENSRNOG00000014135
Rab11fip4
4.607
2.204
0.0453

ENSRNOG00000005052
Slc39a9
4.594
2.200
0.0454

ENSRNOG00000005276
Csnk2a1
4.589
2.198
0.0259

ENSRNOG00000015007
RGD1565591
4.583
2.196
0.0462

ENSRNOG00000002099
Wdfy3
4.579
2.195
0.0217

ENSRNOG00000001747
Pak2
4.572
2.193
0.0178

ENSRNOG00000018226
Zcchc14
4.565
2.190
0.0441

ENSRNOG00000010702
Ube3c
4.564
2.190
0.0154

ENSRNOG00000010610
Hpgd
4.556
2.188
0.0125

ENSRNOG00000001756
D3ZDR3_RAT
4.551
2.186
0.0486

ENSRNOG00000006335
Klhl9
4.550
2.186
0.0083

ENSRNOG00000016715
Kif11
4.547
2.185
0.0159

ENSRNOG00000021916
Slc16a12
4.541
2.183
0.0224

ENSRNOG00000011250
Inmt
4.506
2.172
0.0125

ENSRNOG00000013140
Pdzd2
4.502
2.171
0.0305

ENSRNOG00000012440
Msra
4.501
2.170
0.0308

ENSRNOG00000019932
Ip6k1
4.500
2.170
0.0307

ENSRNOG00000037227
Yes1
4.499
2.170
0.0412

ENSRNOG00000012054
Zmpste24
4.498
2.169
0.0179

ENSRNOG00000007370
Rnf144a
4.493
2.168
0.0443

ENSRNOG00000022968
F1M4Y9_RAT
4.491
2.167
0.0400

ENSRNOG00000011340
D3ZMJ4_RAT
4.488
2.166
0.0143

ENSRNOG00000021705
D3ZXN6_RAT
4.486
2.165
0.0229

ENSRNOG00000003865
Tmigd1
4.483
2.164
0.0072

ENSRNOG00000012105
F1MAE3_RAT
4.478
2.163
0.0346

ENSRNOG00000011312
F1LQ39_RAT
4.475
2.162
0.0366

ENSRNOG00000000127
F1LT58_RAT
4.463
2.158
0.0484

ENSRNOG00000022929
MTMRC_RAT
4.438
2.150
0.0307

ENSRNOG00000033372
Klhl24
4.431
2.148
0.0197

ENSRNOG00000008332
Smo
4.420
2.144
0.0209

ENSRNOG00000028616
Pck1
4.418
2.143
0.0219

ENSRNOG00000013281
Mib1
4.415
2.142
0.0306

ENSRNOG00000011448
Eri1
4.410
2.141
0.0414

ENSRNOG00000028422
Rmnd5a
4.409
2.141
0.0212

ENSRNOG00000014859
Rnf152
4.404
2.139
0.0298

ENSRNOG00000001893
LOC100362453
4.397
2.137
0.0349

ENSRNOG00000018123
Ccny
4.396
2.136
0.0173

ENSRNOG00000016337
Slc22a1
4.394
2.135
0.0356

ENSRNOG00000003709
Kmo
4.389
2.134
0.0166

ENSRNOG00000019939
CCND2_RAT
4.386
2.133
0.0383

ENSRNOG00000029947
—
4.377
2.130
0.0399

ENSRNOG00000008346
Itgb6
4.372
2.128
0.0245

ENSRNOG00000008678
Antxr1
4.357
2.123
0.0237

ENSRNOG00000029924
Klk1l
4.344
2.119
0.0267

ENSRNOG00000043406
LOC100360800
4.341
2.118
0.0323

ENSRNOG00000012343
Pdp2
4.324
2.112
0.0419

ENSRNOG00000009899
D3ZWL1_RAT
4.306
2.106
0.0427

ENSRNOG00000003434
Trove2
4.301
2.105
0.0368

ENSRNOG00000015519
Ces1d
4.294
2.102
0.0253

ENSRNOG00000017439
Cgnl1
4.294
2.102
0.0236

ENSRNOG00000014700
Ttc36
4.287
2.100
0.0266

ENSRNOG00000007944
Edem1
4.281
2.098
0.0367

ENSRNOG00000031263
Haao
4.246
2.086
0.0200

ENSRNOG00000001647
Ets2
4.245
2.086
0.0357

ENSRNOG00000008652
RGD1564964
4.226
2.079
0.0153

ENSRNOG00000023202
Usp15
4.217
2.076
0.0230

ENSRNOG00000016289
Bmpr1b
4.212
2.075
0.0370

ENSRNOG00000015024
E9PT54_RAT
4.208
2.073
0.0252

ENSRNOG00000000555
Eif4ebp2
4.199
2.070
0.0381

ENSRNOG00000008620
Smad3
4.198
2.070
0.0440

ENSRNOG00000008619
Agtrap
4.198
2.070
0.0217

ENSRNOG00000009711
Hepacam2
4.196
2.069
0.0409

ENSRNOG00000015734
Ube3a
4.193
2.068
0.0225

ENSRNOG00000015634
SMAD4_RAT
4.189
2.067
0.0277

ENSRNOG00000042519
RGD1312026
4.182
2.064
0.0380

ENSRNOG00000007564
Evc
4.160
2.057
0.0289

ENSRNOG00000008372
Vamp7
4.160
2.057
0.0433

ENSRNOG00000024671
D4AA13_RAT
4.157
2.056
0.0120

ENSRNOG00000004622
Calcrl
4.142
2.050
0.0131

ENSRNOG00000009660
Enpp6
4.140
2.050
0.0247

ENSRNOG00000014750
D3ZXU7_RAT
4.138
2.049
0.0176

ENSRNOG00000008694
Miox
4.134
2.048
0.0226

ENSRNOG00000004831
Arid2
4.134
2.047
0.0317

ENSRNOG00000043167
Itga9
4.124
2.044
0.0349

ENSRNOG00000001770
Ehhadh
4.114
2.040
0.0104

ENSRNOG00000042160
Tmem167b
4.112
2.040
0.0466

ENSRNOG00000018668
Glg1
4.095
2.034
0.0172

ENSRNOG00000007985
D4ABH6_RAT
4.084
2.030
0.0231

ENSRNOG00000014623
F1M3F2_RAT
4.071
2.026
0.0332

ENSRNOG00000002227
Kit
4.056
2.020
0.0429

ENSRNOG00000016219
Vnn1
4.052
2.019
0.0115

ENSRNOG00000008322
E9PTI4_RAT
4.035
2.013
0.0418

ENSRNOG00000011358
Hipk3
4.034
2.012
0.0372

ENSRNOG00000028335
Fat4
4.017
2.006
0.0190

ENSRNOG00000025554
Zfp445
4.009
2.003
0.0365

ENSRNOG00000003388
Cenpf
3.990
1.996
0.0146

ENSRNOG00000000614
Bicc1
3.987
1.995
0.0162

ENSRNOG00000039091
D3ZRC4_RAT
3.975
1.991
0.0145

ENSRNOG00000030154
Cyp4a2
3.962
1.986
0.0422

ENSRNOG00000033172
—
3.952
1.983
0.0176

ENSRNOG00000017466
Kif5b
3.949
1.981
0.0128

ENSRNOG00000042879
D4A3X0_RAT
3.943
1.979
0.0454

ENSRNOG00000002146
Pkd2
3.942
1.979
0.0358

ENSRNOG00000012940
Vps41
3.937
1.977
0.0280

ENSRNOG00000017291
Sord
3.928
1.974
0.0133

ENSRNOG00000001606
Adamts5
3.923
1.972
0.0420

ENSRNOG00000016534
D3ZKX0_RAT
3.904
1.965
0.0295

ENSRNOG00000007202
Sema3d
3.898
1.963
0.0254

ENSRNOG00000012436
Adh6
3.897
1.962
0.0137

ENSRNOG00000016334
Rod1
3.867
1.951
0.0167

ENSRNOG00000018011
RGD1564456
3.867
1.951
0.0417

ENSRNOG00000039494
D4A608_RAT
3.853
1.946
0.0328

ENSRNOG00000014976
Acsm2
3.850
1.945
0.0330

ENSRNOG00000006636
Otud6b
3.817
1.932
0.0439

ENSRNOG00000015849
Sepp1
3.812
1.930
0.0292

ENSRNOG00000004689
Ptdss1
3.811
1.930
0.0342

ENSRNOG00000013808
Ces2g
3.803
1.927
0.0345

ENSRNOG00000014673
Eri2
3.791
1.922
0.0429

ENSRNOG00000009819
Vezf1
3.784
1.920
0.0450

ENSRNOG00000016758
Loxl2
3.783
1.919
0.0310

ENSRNOG00000010061
Gmfb
3.763
1.912
0.0466

ENSRNOG00000023021
Msl2
3.746
1.906
0.0486

ENSRNOG00000039571
Glod5
3.742
1.904
0.0370

ENSRNOG00000017600
Ptpn9
3.739
1.903
0.0329

ENSRNOG00000000590
Naglt1
3.718
1.895
0.0365

ENSRNOG00000011511
Stk24
3.716
1.894
0.0398

ENSRNOG00000018279
Sfxn1
3.712
1.892
0.0213

ENSRNOG00000003953
RB3GP_RAT
3.709
1.891
0.0486

ENSRNOG00000024632
Atf6
3.699
1.887
0.0421

ENSRNOG00000016779
Fam120a
3.679
1.879
0.0257

ENSRNOG00000010379
Cugbp1
3.670
1.876
0.0295

ENSRNOG00000010780
Dlc1
3.664
1.874
0.0495

ENSRNOG00000003948
Llgl1
3.659
1.871
0.0486

ENSRNOG00000016183
Ipp
3.647
1.867
0.0466

ENSRNOG00000017964
Slc22a25
3.567
1.835
0.0201

ENSRNOG00000039745
Pm20d1
3.557
1.831
0.0300

ENSRNOG00000010107
PALLD_RAT
3.528
1.819
0.0467

ENSRNOG00000019444
D4ADJ6_RAT
3.515
1.814
0.0265

ENSRNOG00000011260
Cmbl
3.513
1.813
0.0221

ENSRNOG00000013322
DPOLA_RAT
3.505
1.810
0.0498

ENSRNOG00000039278
Mcart1
3.477
1.798
0.0345

ENSRNOG00000021108
Slc22a12
3.449
1.786
0.0263

ENSRNOG00000010887
RGD1309534
3.435
1.781
0.0382

ENSRNOG00000008331
RGD1309995
3.394
1.763
0.0425

ENSRNOG00000007949
Rgn
3.355
1.746
0.0276

ENSRNOG00000011987
Cd2ap
3.343
1.741
0.0306

ENSRNOG00000042175
B6VQA7_RAT
3.331
1.736
0.0384

ENSRNOG00000012190
Cldn2
3.324
1.733
0.0347

ENSRNOG00000023972
F1M6Q3_RAT
3.323
1.733
0.0322

ENSRNOG00000011763
Serp1
3.319
1.731
0.0316

ENSRNOG00000004496
Rock2
3.318
1.730
0.0337

ENSRNOG00000004677
Zeb2
3.306
1.725
0.0306

ENSRNOG00000013409
Gclm
3.301
1.723
0.0338

ENSRNOG00000004302
Pah
3.270
1.709
0.0368

ENSRNOG00000010947
MMP14_RAT
3.253
1.702
0.0337

ENSRNOG00000011058
Utrn
3.247
1.699
0.0378

ENSRNOG00000018215
Slc22a6
3.246
1.698
0.0377

ENSRNOG00000016456
Il33
3.234
1.693
0.0319

ENSRNOG00000002541
Pds5a
3.164
1.662
0.0449

ENSRNOG00000002680
Lamc1
3.139
1.650
0.0366

ENSRNOG00000011124
Eif4g2-ps1
3.125
1.644
0.0380

ENSRNOG00000004009
Xpnpep2
3.118
1.641
0.0414

ENSRNOG00000010768
Kpna4
3.114
1.639
0.0499

ENSRNOG00000042249
F1LTA7_RAT
3.101
1.633
0.0422

ENSRNOG00000014166
Smoc2
3.078
1.622
0.0413

ENSRNOG00000002305
Slc15a2
3.061
1.614
0.0400

ENSRNOG00000005130
Ogdh
3.049
1.608
0.0465

ENSRNOG00000018086
Slc22a8
3.048
1.608
0.0418

ENSRNOG00000010814
Bmpr1a
3.006
1.588
0.0432

ENSRNOG00000032885
CYC_RAT
2.936
1.554
0.0478

Down-regulated Genes: 15

ENSRNOG00000032087
F1LWC2_RAT
0.301
−1.734
0.0328

ENSRNOG00000032609
—
0.300
−1.738
0.0394

ENSRNOG00000033748
F1LWC2_RAT
0.299
−1.741
0.0381

ENSRNOG00000025670
Shisa3
0.295
−1.759
0.0282

ENSRNOG00000029115
—
0.284
−1.815
0.0315

ENSRNOG00000011821
S100a4
0.228
−2.134
0.0086

ENSRNOG00000007632
Zmynd17
0.211
−2.243
0.0365

ENSRNOG00000025408
D3ZTT0_RAT
0.189
−2.403
0.0422

ENSRNOG00000028844
Slc9a5
0.181
−2.466
0.0282

ENSRNOG00000006889
Ambp
0.150
−2.741
0.0177

ENSRNOG00000028730
D3ZI71_RAT
0.148
−2.757
0.0450

ENSRNOG00000026067
Wfdc10
0.144
−2.791
0.0123

ENSRNOG00000037374
D3ZPQ1_RAT
0.140
−2.840
0.0087

ENSRNOG00000033517
LOC100360791
0.129
−2.950
0.0009

ENSRNOG00000042909
F1LZX4_RAT
0.107
−3.229
0.0466

ENSRNOG00000014578
Fxyd4
0.096
−3.374
0.0001

Figure 6: Enzymes of the tryptophan metabolism pathway that are regulated by melatonin therapy in the kidney (red stars). Data were analyzed using the KEGG Pathway feature of the DAVID software.

4. Discussion

The major findings of our study can be summarized as follows: (1) CR offspring developed hypertension at 12 weeks of age and this was prevented by maternal melatonin therapy; (2) melatonin restored the CR-induced increase of plasma ADMA level, decreased L-arginine level, and decreased L-arginine-to-ADMA ratio; (3) CR reduced renal NO level and this was prevented by melatonin; (4) melatonin therapy increased PAX2 mRNA expression in the CR + M group; (5) CR upregulated renin and PRR expression and melatonin suppressed this increase; (6) melatonin therapy significantly increased renal ACE2 protein levels in the M and CR + M group; and (7) the expression of numerous genes was regulated in melatonin-treated offspring kidneys during nephrogenesis.

Our recent work indicates that ADMA-induced NO/reactive oxygen species (ROS) imbalance is involved in the development of hypertension in two different developmental models, maternal caloric restriction, and maternal diabetes [10, 11]. Several lines of evidence in this study indicated that ADMA-induced NO/ROS imbalance is involved in the developmental programming of hypertension in offspring exposed to maternal caloric restriction. First, plasma levels of the endogenous NOS inhibitor ADMA were increased in the CR group. Second, ADMA and L-arginine both compete for NOS and are present in a ratio that maintains NO homeostasis; this ratio was decreased in the plasma in the CR group. Third, maternal CR decreased renal NO levels in the offspring. Thus, alterations in the ADMA-NO pathway might be a major factor involved in programmed adult hypertension in response to maternal CR.

Melatonin is rapidly transferred from maternal to fetal circulation [20]. Administration of melatonin to pregnant rats prevents oxidative stress damage in the brains of offspring [21]. Previously, we showed that melatonin increases NO, restoring NO/ROS balance at the prehypertension stage and leading to lower blood pressure in young SHR [15]. Consistent with these findings, we found that early melatonin therapy in the mother could prevent programmed hypertension in their adult offspring. Thus, we suggest that melatonin has a novel protective effect on programmed hypertension through acting on the ADMA-NO pathway.

In addition to oxidative stress, the RAS plays a fundamental role in the development of hypertension and kidney development [5]. Epigenetic regulation of several RAS components has been reported in different programmed hypertension models [22, 23]. We demonstrated for the first time that melatonin therapy during nephrogenesis increased renin, PRR, and ACE2 expression in the kidney of the adult offspring. Consistent with these data, renal protein levels of PRR and ACE2 were increased in melatonin-treated offspring. Renin-PRR signaling is essential for proper kidney development and is causally linked to hypertension [13]. ACE2 appears to antagonize the effects of ACE through the production of angiotensin (1–7) in a manner that opposes the development of hypertension [24]. Surprisingly, melatonin therapy increased ACE2 expression in the kidney and prevented CR-induced programmed hypertension, despite the presence of increased renin and PRR expression. Notably, melatonin upregulated several RAS components and had reciprocal effects on vasodilation and vasoconstriction in rats at 3 months of age. Future studies are required to clarify the underlying mechanisms involved in the differential regulation of RAS components by melatonin.

Long-term amelioration of hypertension by melatonin therapy during gestation and lactation may be due to epigenetic changes in the kidney during a critical period of nephrogenesis. We found that melatonin upregulated HDAC-2, -3, and -8 expression in the kidney in CR + M group. This finding is consistent with that of our previous study showing that melatonin increased the expression of both class I and class II HDACs in vitro [25]. Given that melatonin increased class I HDACs expression and that HDACs are primarily thought to repress gene transcription, melatonin likely upregulates gene expression. Conversely, melatonin is known as a class III HDAC inhibitor [17]. Thus, melatonin might have dual effects on HDACs to epigenetically regulate gene expression. To the best of our knowledge, our study is the first to document altered expression of more than 400 genes in the kidney in response to melatonin and implicates melatonin in the protection from programmed hypertension in adult life. Notably, our data imply that melatonin is liable to induce, but not suppress, gene expression in the developing kidney. Using the KEGG database, several biological pathways were proposed to be regulated by melatonin including focal adhesion signaling, the peroxisome proliferator-activated receptors signaling pathway, fatty acid metabolism, the transforming growth factor signaling pathway, and the Wnt signaling pathway. These findings suggest that melatonin might have a global epigenetic effect during nephrogenesis. Interestingly, the most significantly regulated biological theme was tryptophan metabolism, indicating that melatonin might have a negative feedback effect on its precursor tryptophan. Notably, maternal melatonin therapy has adverse effects on survival and renal growth in Wistar-Kyoto rats [26]. Because our data showed that maternal melatonin therapy had strong epigenetic effects, further evaluation is warranted to determine whether early melatonin therapy causes long-term epigenetic changes that lead to adverse effects in adulthood.

Previously, we showed that maternal CR reduces nephron numbers in offspring [10]. Increases in renal apoptosis and impaired expression of nephrogenesis-related genes may contribute to this reduction. In contrast to several earlier reports [27, 28], we found that apoptosis- and nephrogenesis-related genes were not altered in maternal CR-induced programmed hypertension. Of note, we showed for the first time that melatonin treatment upregulated PAX2 mRNA in metanephroi. Because PAX2 plays a crucial role in kidney development and is associated with various congenital renal and ureteral malformations, further studies are warranted to understand the epigenetic regulation of melatonin on PAX2 during nephrogenesis.

We conclude that prenatal melatonin therapy offsets the effects of maternal CR-induced programmed hypertension in adult offspring, primarily through the restoration of the ADMA-NO balance in the kidney. Our data suggested that a critical window exists during nephrogenesis in which the adult BP can be modified. Moreover, we showed that melatonin can modulate type I HDACs and serve as an inducer of gene expression in the developing kidney. The implications of melatonin-induced epigenetic changes on programmed hypertension in later life remain to be explored.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by Grant NSC 101-2314-B-182A-021-MY3 from the National Science Council (Taiwan) and Grants CMRPG8B0172 and CMRPG8C0041 from Chang Gung Memorial Hospital (Kaohsiung, Taiwan).

References

N. B. Ojeda, D. Grigore, and B. T. Alexander, “Developmental programming of hypertension: insight from animal models of nutritional manipulation,” Hypertension, vol. 52, no. 1, pp. 44–50, 2008.

S. P. Bagby, “Maternal nutrition, low nephron number, and hypertension in later life: pathways of nutritional programming,” Journal of Nutrition, vol. 137, no. 4, pp. 1066–1072, 2007.

I. C. McMillen and J. S. Robinson, “Developmental origins of the metabolic syndrome: prediction, plasticity, and programming,” Physiological Reviews, vol. 85, no. 2, pp. 571–633, 2005.

S. H. Alwasel and N. Ashton, “Prenatal programming of renal sodium handling in the rat,” Clinical Science, vol. 117, no. 2, pp. 75–84, 2009.

V. M. Vehaskari, T. Stewart, D. Lafont, C. Soyez, D. Seth, and J. Manning, “Kidney angiotensin and angiotensin receptor expression in prenatally programmed hypertension,” The American Journal of Physiology—Renal Physiology, vol. 287, no. 2, pp. F262–F267, 2004.

P. A. Dennery, “Oxidative stress in development: nature or nurture?” Free Radical Biology and Medicine, vol. 49, no. 7, pp. 1147–1151, 2010.

C. S. Wilcox, “Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension?” The American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 289, no. 4, pp. R913–R935, 2005.

C. Baylis, “Arginine, arginine analogs and nitric oxide production in chronic kidney disease,” Nature Clinical Practice Nephrology, vol. 2, no. 4, pp. 209–220, 2006.

Y.-L. Tain and L.-T. Huang, “Asymmetric dimethylarginine: clinical applications in pediatric medicine,” Journal of the Formosan Medical Association, vol. 110, no. 2, pp. 70–77, 2011.

Y.-L. Tain, C.-S. Hsieh, I.-C. Lin, C.-C. Chen, J.-M. Sheen, and L.-T. Huang, “Effects of maternal l-citrulline supplementation on renal function and blood pressure in offspring exposed to maternal caloric restriction: the impact of nitric oxide pathway,” Nitric Oxide, vol. 23, no. 1, pp. 34–41, 2010.

Y. L. Tain, W. C. Lee, C. N. Hsu et al., “Asymmetric dimethylarginine is associated with developmental programming of adult kidney disease and hypertension in offspring of streptozotocin-treated mothers,” PLoS ONE, vol. 8, no. 2, Article ID e55420, 2013.

S. Chen, C. Bellew, X. Yao et al., “Histone deacetylase (HDAC) activity is critical for embryonic kidney gene expression, growth, and differentiation,” The Journal of Biological Chemistry, vol. 286, no. 37, pp. 32775–32789, 2011.

R. Song, T. Van Buren, and I. V. Yosypiv, “Histone deacetylases are critical regulators of the renin-angiotensin system during ureteric bud branching morphogenesis,” Pediatric Research, vol. 67, no. 6, pp. 573–578, 2010.

A. Galano, D. X. Tan, and R. J. Reiter, “On the free radical scavenging activities of melatonin's metabolites, AFMK and AMK,” Journal of Pineal Research, vol. 54, no. 3, pp. 245–257, 2013.

Y.-L. Tain, L.-T. Huang, I.-C. Lin, Y.-T. Lau, and C.-Y. Lin, “Melatonin prevents hypertension and increased asymmetric dimethylarginine in young spontaneous hypertensive rats,” Journal of Pineal Research, vol. 49, no. 4, pp. 390–398, 2010.

A. Korkmaz, S. Rosales-Corral, and R. J. Reiter, “Gene regulation by melatonin linked to epigenetic phenomena,” Gene, vol. 503, no. 1, pp. 1–11, 2012.

B. Jung-Hynes, R. J. Reiter, and N. Ahmad, “Sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer,” Journal of Pineal Research, vol. 48, no. 1, pp. 9–19, 2010.

Y.-L. Tain and C. Baylis, “Determination of dimethylarginine dimethylaminohydrolase activity in the kidney,” Kidney International, vol. 72, no. 7, pp. 886–889, 2007.

A. Mortazavi, B. A. Williams, K. McCue, L. Schaeffer, and B. Wold, “Mapping and quantifying mammalian transcriptomes by RNA-Seq,” Nature Methods, vol. 5, no. 7, pp. 621–628, 2008.

Y. Okatani, A. Wakatsuki, and C. Kaneda, “Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain,” Journal of Pineal Research, vol. 28, no. 2, pp. 89–96, 2000.

K. Watanabe, A. Wakatsuki, K. Shinohara, N. Ikenoue, K. Yokota, and T. Fukaya, “Maternally administered melatonin protects against ischemia and reperfusion-induced oxidative mitochondrial damage in premature fetal rat brain,” Journal of Pineal Research, vol. 37, no. 4, pp. 276–280, 2004.

I. Bogdarina, S. Welham, P. J. King, S. P. Burns, and A. J. L. Clark, “Epigenetic modification of the renin-angiotensin system in the fetal programming of hypertension,” Circulation Research, vol. 100, no. 4, pp. 520–526, 2007.

R. Goyal, D. Goyal, A. Leitzke, C. P. Gheorghe, and L. D. Longo, “Brain renin-angiotensin system: fetal epigenetic programming by maternal protein restriction during pregnancy,” Reproductive Sciences, vol. 17, no. 3, pp. 227–238, 2010.

Y. Feng, H. Xia, R. A. Santos, R. Speth, and E. Lazartigues, “Angiotensin-converting enzyme 2: a new target for neurogenic hypertension,” Experimental Physiology, vol. 95, no. 5, pp. 601–606, 2010.

R. Sharma, T. Ottenhof, P. A. Rzeczkowska, and L. P. Niles, “Epigenetic targets for melatonin: induction of histone H3 hyperacetylation and gene expression in C17.2 neural stem cells,” Journal of Pineal Research, vol. 45, no. 3, pp. 277–284, 2008.

H. J. Singh, L. S. Keah, A. Kumar, and K. N. S. Sirajudeen, “Adverse effects of melatonin on rat pups of Wistar-Kyoto dams receiving melatonin supplementation during pregnancy,” Experimental and Toxicologic Pathology, vol. 64, no. 7-8, pp. 751–752, 2012.

H. Dickinson, D. W. Walker, E. M. Wintour, and K. Moritz, “Maternal dexamethasone treatment at midgestation reduces nephron number and alters renal gene expression in the fetal spiny mouse,” The American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 292, no. 1, pp. R453–R461, 2007.

A. K. Abdel-Hakeem, T. Q. Henry, T. R. Magee et al., “Mechanisms of impaired nephrogenesis with fetal growth restriction: altered renal transcription and growth factor expression,” The American Journal of Obstetrics and Gynecology, vol. 199, no. 3, pp. 252.e1–252.e7, 2008.