Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 283180 | 21 pages | https://doi.org/10.1155/2014/283180

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

Academic Editor: Jean-Claude Lavoie
Received06 Jan 2014
Revised18 Mar 2014
Accepted28 Mar 2014
Published22 Apr 2014

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 [25].

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 .


GeneForwardReverse

Bax5 ttgctgatggcaacttcaactg 35 ctttagtgcacagggccttgag 3
P535 catgagcgttgctctgatg 35 cagatactcagcatacggatttcc 3
PAX25 gagactcccagagtggtgtg 35 cattcccctgttctgatttg 3
FGF25 ccagttggtatgtggcactg 35 cagggaagggtttgacaaga 3
BMP45 gacttcgaggcgacacttctg 35 agccggtaaagatccctcatg 3
Renin5 aacattaccagggcaactttcact 35 acccccttcatggtgatctg 3
Prorenin receptor5 gaggcagtgaccctcaacat 35 ccctcctcacacaacaaggt 3
Angiotensinogen5 gcccaggtcgcgatgat 35 tgtacaagatgctgagtgaggcaa 3
ACE5 caccggcaaggtctgctt 35 cttggcatagtttcgtgaggaa 3
ACE25 acccttcttacatcagccctactg 35 tgtccaaaacctaccccacatat 3
AT1R5 gctgggcaacgagtttgtct 35 cagtccttcagctggatcttca 3
AT2R5 caatctggctgtggctgactt 35 tgcacatcacaggtccaaaga 3
MAS5 catctctcctctcggctttgtg 35 cctcatccggaagcaaagg 3
HDAC-15 gaactggggacctacggg 3 5 gctcttgacaaattccacacac 3
HDAC-25 agttgcccttgattgtgaga 3 5 ccactgttgtccttggatttat 3
HDAC-35 tgatgaccagagttacaagcac 3 5 gggcaacatttc ggacag 3
HDAC-85 gctacccccggtttatatttacag 3 5 ttcgatcagagagtgaaccatactg 3
R18S5 gccgcggtaattccagctcca 35 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.


 ControlCRMCR + M

Mortality10%0%0%0%
Body weight (BW) (g)435 ± 14356 ± 4*
Left kidney weight (g)1.22 ± 0.061.01 ± 0.021.97 ± 0.05*#1.48 ± 0.03#$
Left kidney weight/100g BW0.28 ± 0.010.28 ± 0.010.4 ± 0.01*#0.4 ± 0.01*#
Heart weight (g)1.23 ± 0.051.24 ± 0.021.63 ± 0.01*#1.16 ± 0.05$
Heart weight/100 g BW0.28 ± 0.010.35 ± 0.01*0.35 ± 0.01*0.31 ± 0.01
Systolic blood pressure (mmHg)162 ± 2180 ± 2*155 ± 1#166 ± 1$
Diastolic blood pressure (mmHg)122 ± 2134 ± 3*108 ± 2*#113 ± 1*#
Mean arterial pressure (mmHg)135 ± 2149 ± 2*124 ± 1*#131 ± 1#$
CCr, mL·min−1·kg body weight−19.12 ± 3.458.5 ± 3.07.34 ± 2.327.81 ± 2.76

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


Control CRMCR + M

Plasma (μmol)
 L-Citrulline 50 ± 4.161 ± 3.659.3 ± 5.155.8 ± 6.9
 L-Arginine 121.1 ± 1484.4 ± 2.4*113.6 ± 8.7#112.8 ± 13.6#
 ADMA 1.31 ± 0.12.21 ± 0.18*1.18 ± 0.06#1.08 ± 0.12#
 SDMA 0.66 ± 0.041.62 ± 0.27*0.97 ± 0.09#0.92 ± 0.08#
 L-Arginine-to-ADMA ratio92 ± 840 ± 4*98 ± 10#105 ± 6#
Kidney (μmol/mg protein)
 L-Citrulline 52.5 ± 8.653.1 ± 4.697.6 ± 8.468.8 ± 12.4
 L-Arginine 425 ± 62.3552.9 ± 58.9522.8 ± 61.6488.1 ± 56
 ADMA 5.09 ± 0.886.33 ± 0.716.72 ± 1.034.84 ± 0.61
 SDMA 4.3 ± 0.655.3 ± 0.515.57 ± 0.794.59 ± 0.73
 L-Arginine-to-ADMA ratio86 ± 489 ± 580 ± 4103 ± 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.

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)).

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)).

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.

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).


Gene_IDGene symbolFold changesLog2 value

Upregulated: 439 genes
ENSRNOG00000038989D3ZSD6_RAT28.6864.8420.0083
ENSRNOG00000006367Slc5a819.2644.2680.0001
ENSRNOG00000003038Sft2d217.1014.0960.0020
ENSRNOG00000007720F1LX97_RAT13.8413.7910.0027
ENSRNOG00000019014Ndst112.6573.6620.0003
ENSRNOG00000017434Mgat312.3643.6280.0005
ENSRNOG00000001656Kcnj1511.7243.5510.0028
ENSRNOG0000002129211.4493.5170.0015
ENSRNOG00000017078Sepn111.1073.4730.0011
ENSRNOG00000030121Enpep10.9703.4560.0029
ENSRNOG00000005854Angpt110.9203.4490.0165
ENSRNOG00000009944LOC31440710.8583.4410.0017
ENSRNOG00000013279Scd10.7753.4300.0012
ENSRNOG00000001724LOC67870410.6903.4180.0011
ENSRNOG00000002463LOC68275210.6333.4110.0033
ENSRNOG00000011630Ak3l110.3043.3650.0441
ENSRNOG00000005447RGD131156410.1173.3390.0026
ENSRNOG00000009019Slc6a610.0903.3350.0016
ENSRNOG00000002969Itpkb9.8923.3060.0020
ENSRNOG00000037307Spata229.8763.3040.0036
ENSRNOG00000039717Ipo119.5843.2610.0069
ENSRNOG00000025372Glce9.5363.2530.0023
ENSRNOG00000037884Oxgr19.5103.2490.0169
ENSRNOG00000021203Atl39.4873.2460.0056
ENSRNOG00000006787Dhcr249.3283.2220.0023
ENSRNOG00000015038Adam109.2793.2140.0005
ENSRNOG00000002519Magt19.2533.2100.0010
ENSRNOG00000038933D3ZF12_RAT9.2253.2060.0023
ENSRNOG00000024757RGD13104449.1193.1890.0066
ENSRNOG00000030285Epha39.0643.1800.0032
ENSRNOG00000018338Vwa19.0173.1730.0355
ENSRNOG00000022802Tmem184b8.9823.1670.0070
ENSRNOG00000013265Tgfbr28.9473.1610.0014
ENSRNOG00000026941Tril8.9343.1590.0024
ENSRNOG00000020532Kcnq18.9043.1540.0441
ENSRNOG00000018503LOC2931908.8623.1480.0172
ENSRNOG00000002198LOC6853528.7113.1230.0037
ENSRNOG00000017172Fam125b8.7063.1220.0291
ENSRNOG000000185548.6633.1150.0067
ENSRNOG00000013963IL6RB_RAT8.5713.0990.0023
ENSRNOG000000425658.5473.0950.0114
ENSRNOG00000032834Hspa138.5443.0950.0011
ENSRNOG00000002355Slc47a18.4743.0830.0025
ENSRNOG00000011927SDC3_RAT8.4603.0810.0065
ENSRNOG00000042540Mef2a8.4543.0800.0368
ENSRNOG00000029216Dgcr28.3313.0590.0175
ENSRNOG00000023725LOC6897568.2153.0380.0191
ENSRNOG00000028129Fktn8.2073.0370.0063
ENSRNOG00000000547Tspyl48.2023.0360.0103
ENSRNOG00000011859Eif5a28.1923.0340.0403
ENSRNOG00000028387E9PTK5_RAT8.1683.0300.0197
ENSRNOG00000015986Rassf88.1343.0240.0094
ENSRNOG00000029409Gstm6l8.0623.0110.0292
ENSRNOG00000008895Hnf4a7.9932.9990.0398
ENSRNOG00000038149Defb97.9482.9910.0437
ENSRNOG00000040287Cyp1b17.8902.9800.0439
ENSRNOG00000010468Elovl67.8712.9770.0394
ENSRNOG00000014524F1M9D3_RAT7.8282.9690.0077
ENSRNOG00000014209Utp67.7932.9620.0050
ENSRNOG00000013419Agphd17.7892.9610.0031
ENSRNOG00000020653S1pr27.7752.9590.0313
ENSRNOG00000018714Arl5b7.7702.9580.0078
ENSRNOG00000002408Rbm477.7192.9480.0057
ENSRNOG00000008971Hnf4g7.7152.9480.0085
ENSRNOG00000011271Mcc7.6882.9430.0120
ENSRNOG00000002276LOC1003597147.6412.9340.0073
ENSRNOG00000009446Rxra7.6072.9270.0066
ENSRNOG00000019400Dag17.5912.9240.0010
ENSRNOG00000013098F1M9J1_RAT7.5812.9220.0320
ENSRNOG00000014511Alg107.5512.9170.0076
ENSRNOG00000012490Amph7.5332.9130.0461
ENSRNOG00000014934Fam63b7.4812.9030.0193
ENSRNOG00000039630LOC2905777.4142.8900.0045
ENSRNOG00000032707Egf7.3682.8810.0017
ENSRNOG00000015605Ptprk7.3572.8790.0298
ENSRNOG00000000168Gatm7.3112.8700.0017
ENSRNOG00000027097F1M683_RAT7.2732.8630.0087
ENSRNOG00000018109Clic47.2512.8580.0048
ENSRNOG00000008629Secisbp2l7.2362.8550.0042
ENSRNOG00000019799Pcdhgc37.2312.8540.0246
ENSRNOG00000024089Fndc3b7.2212.8520.0065
ENSRNOG00000015852D4AD82_RAT7.1922.8460.0020
ENSRNOG00000006967Xiap7.1512.8380.0136
ENSRNOG00000031487F1LM52_RAT7.1292.8340.0477
ENSRNOG00000014866Pign7.0772.8230.0190
ENSRNOG00000033206Entpd57.0602.8200.0070
ENSRNOG00000037753Slc10a27.0022.8080.0089
ENSRNOG00000040195F1LZT0_RAT7.0012.8080.0018
ENSRNOG00000042817D4A5M8_RAT6.9252.7920.0104
ENSRNOG00000005070Spopl6.9202.7910.0139
ENSRNOG00000006459D4AEA4_RAT6.8712.7810.0251
ENSRNOG00000012784Gtf3c46.8502.7760.0096
ENSRNOG00000016968Gramd46.8382.7740.0216
ENSRNOG00000004448RGD13070516.8192.7700.0050
ENSRNOG00000021809Gpx36.8012.7660.0008
ENSRNOG00000014183Gnaq6.8012.7660.0084
ENSRNOG00000012991LOC1003632756.7982.7650.0046
ENSRNOG00000013443Tm9sf36.7912.7640.0040
ENSRNOG00000042673LOC1003595446.7892.7630.0012
ENSRNOG00000003873Cpd6.7672.7580.0028
ENSRNOG00000007990Adipor26.7622.7580.0026
ENSRNOG00000007804C1galt16.7622.7570.0109
ENSRNOG00000043256D3ZNR8_RAT6.7202.7490.0145
ENSRNOG00000015124Gpam6.7202.7480.0109
ENSRNOG00000004888Spred26.6902.7420.0454
ENSRNOG00000003960Tmem276.6822.7400.0026
ENSRNOG00000015750Wnt7b6.6542.7340.0218
ENSRNOG00000030763Dpp46.6012.7230.0011
ENSRNOG00000039504Q5M885_RAT6.5622.7140.0116
ENSRNOG00000032768D3Z9G8_RAT6.4972.7000.0214
ENSRNOG00000039771LOC1003616296.4942.6990.0140
ENSRNOG00000009274Fut116.4752.6950.0354
ENSRNOG00000027938RGD15620376.4202.6830.0117
ENSRNOG00000001335Zkscan16.4192.6820.0077
ENSRNOG00000004978Prkacb6.3792.6730.0216
ENSRNOG00000005446Gna116.3632.6700.0172
ENSRNOG00000003884Acmsd6.3622.6690.0262
ENSRNOG00000028190D4ACF8_RAT6.3542.6680.0432
ENSRNOG00000006338Lrp66.3512.6670.0041
ENSRNOG00000009523Rab11fip26.3452.6660.0470
ENSRNOG00000003759Galc6.3452.6660.0140
ENSRNOG00000010620NDC1_RAT6.3192.6600.0275
ENSRNOG00000001821Adipoq6.3062.6570.0244
ENSRNOG00000038572RGD15626466.2932.6540.0106
ENSRNOG00000026120Fam8a16.2822.6510.0129
ENSRNOG00000025476RGD13593496.2432.6420.0126
ENSRNOG00000019508Wars26.2162.6360.0317
ENSRNOG00000008271Fam91a16.2162.6360.0031
ENSRNOG00000017120Abhd26.2082.6340.0278
ENSRNOG00000010843Nhlrc36.2032.6330.0255
ENSRNOG00000030704F1LV74_RAT6.1392.6180.0369
ENSRNOG00000002509Gnl3l6.1292.6160.0124
ENSRNOG00000010841Col8a26.0892.6060.0457
ENSRNOG00000002728Btc6.0882.6060.0348
ENSRNOG00000027320Eif2c16.0822.6050.0243
ENSRNOG00000009453Mobkl2b6.0722.6020.0271
ENSRNOG00000007797Rbpsuh6.0692.6020.0133
ENSRNOG00000017286HYES_RAT6.0642.6000.0032
ENSRNOG00000002461Nid16.0572.5990.0014
ENSRNOG00000006649Thrb6.0482.5960.0180
ENSRNOG00000025042Pde3a6.0482.5960.0189
ENSRNOG00000015916Ttc386.0482.5960.0384
ENSRNOG00000013581Extl36.0382.5940.0093
ENSRNOG00000002332MSPD1_RAT6.0342.5930.0213
ENSRNOG00000032757D3Z903_RAT6.0322.5930.0431
ENSRNOG00000029651Rdh26.0252.5910.0258
ENSRNOG00000018588Sox46.0192.5900.0342
ENSRNOG00000012428Maf6.0052.5860.0483
ENSRNOG00000009506Mre11a6.0052.5860.0332
ENSRNOG000000283305.9872.5820.0375
ENSRNOG00000034025D4A4T5_RAT5.9792.5800.0227
ENSRNOG00000007079Met5.9792.5800.0117
ENSRNOG00000008088Btbd35.9792.5800.0181
ENSRNOG00000017546Mylk35.9452.5720.0224
ENSRNOG00000042333Dnal15.8952.5590.0187
ENSRNOG00000001092Kl5.8732.5540.0106
ENSRNOG000000164985.8362.5450.0049
ENSRNOG00000037765Lims15.8332.5440.0367
ENSRNOG00000010267Klhdc105.8272.5430.0346
ENSRNOG00000043277D3ZIC7_RAT5.8092.5380.0206
ENSRNOG00000024799D3ZNV9_RAT5.8032.5370.0032
ENSRNOG00000004919Gns5.7952.5350.0282
ENSRNOG00000015080Wdfy15.7662.5280.0292
ENSRNOG00000009565Pdk45.7642.5270.0206
ENSRNOG00000013082LCAP_RAT5.7542.5250.0322
ENSRNOG00000026501Slc6a195.7422.5220.0406
ENSRNOG00000009597Cyp4a15.7402.5210.0123
ENSRNOG00000011560Mtmr95.7382.5210.0368
ENSRNOG00000022710Prrg45.7362.5200.0269
ENSRNOG00000013469LOC1003628055.7152.5150.0059
ENSRNOG00000024640RGD13047315.6982.5100.0080
ENSRNOG00000018952Sema3g5.6922.5090.0143
ENSRNOG00000020011Q66HF5_RAT5.6772.5050.0381
ENSRNOG00000012826Creb3l25.6652.5020.0189
ENSRNOG00000032492Usp225.6572.5000.0107
ENSRNOG00000021840LOC5000465.6442.4970.0118
ENSRNOG00000034026Lclat15.6422.4960.0223
ENSRNOG00000009153Cidec5.6422.4960.0432
ENSRNOG00000028899Zbtb335.6332.4940.0168
ENSRNOG00000001766Tfrc5.6132.4890.0102
ENSRNOG00000017901Acy35.6132.4890.0044
ENSRNOG00000012095Pkia5.5962.4840.0339
ENSRNOG00000001796Dgkg5.5732.4790.0471
ENSRNOG00000004958RGD13046055.5632.4760.0100
ENSRNOG00000025587Plagl15.5502.4720.0289
ENSRNOG00000027540Fam102b5.5362.4690.0410
ENSRNOG00000001518Itga65.5192.4650.0452
ENSRNOG00000032723Eftud15.5152.4630.0336
ENSRNOG00000002053F1M3H3_RAT5.4912.4570.0081
ENSRNOG00000003472Atp11c-ps15.4732.4520.0317
ENSRNOG00000003984Apln5.4482.4460.0337
ENSRNOG00000012453RGD15645605.4382.4430.0046
ENSRNOG00000017846Slc44a15.4222.4390.0293
ENSRNOG00000016921Klhl115.4182.4380.0275
ENSRNOG00000026415D4A301_RAT5.4032.4340.0280
ENSRNOG00000013798Fnbp1l5.3912.4310.0098
ENSRNOG00000003620Fmo35.3842.4290.0050
ENSRNOG00000018220Pde4dip5.3772.4270.0462
ENSRNOG00000000145Pik3r35.3522.4200.0210
ENSRNOG00000008834LOC3060965.3512.4200.0356
ENSRNOG00000025882Nipal15.3452.4180.0306
ENSRNOG00000010996Mobkl1a5.3412.4170.0147
ENSRNOG00000001582Bach15.3392.4170.0199
ENSRNOG00000022309D3ZRU8_RAT5.3132.4100.0048
ENSRNOG00000015741Slc2a135.2982.4060.0371
ENSRNOG00000014303F1M753_RAT5.2942.4040.0391
ENSRNOG00000036798Dusp35.2842.4020.0199
ENSRNOG00000012142Glyat5.2832.4010.0081
ENSRNOG00000024426D3ZXW1_RAT5.2592.3950.0477
ENSRNOG00000006628Dusp165.2562.3940.0271
ENSRNOG00000026143Ckap2l5.2302.3870.0271
ENSRNOG00000018867Klhdc7a5.2232.3850.0489
ENSRNOG00000025296Lrrc8a5.2032.3790.0176
ENSRNOG00000014508Mgll5.2032.3790.0137
ENSRNOG00000000589RGD13104955.1992.3780.0372
ENSRNOG00000014234Hif1an5.1922.3760.0394
ENSRNOG00000008450LOC1003595395.1782.3720.0409
ENSRNOG00000010744Nrp15.1772.3720.0072
ENSRNOG00000039837RGD15639455.1612.3680.0466
ENSRNOG00000013177Map3k15.1542.3660.0114
ENSRNOG00000021719F1LX81_RAT5.1532.3650.0133
ENSRNOG00000024629Hadha5.1162.3550.0126
ENSRNOG00000014907Aldh8a15.1052.3520.0055
ENSRNOG00000036673Sectm1b5.0982.3500.0121
ENSRNOG00000024794Senp55.0962.3490.0264
ENSRNOG00000005131Lin7c5.0862.3470.0289
ENSRNOG00000002225Scarb25.0812.3450.0116
ENSRNOG00000020284Prkar2a5.0772.3440.0215
ENSRNOG00000014648Efnb25.0722.3430.0303
ENSRNOG00000002488Galnt105.0632.3400.0437
ENSRNOG00000017406Atrnl15.0562.3380.0269
ENSRNOG00000010813Tspan145.0482.3360.0304
ENSRNOG00000000645Reep35.0472.3360.0262
ENSRNOG00000018873Fam168a5.0362.3320.0160
ENSRNOG00000020253RAB1B_RAT5.0302.3310.0128
ENSRNOG00000001235Gna125.0122.3250.0149
ENSRNOG00000040215F1LZL1_RAT5.0112.3250.0302
ENSRNOG00000011619Myo9a4.9882.3190.0163
ENSRNOG00000039976D3ZHG3_RAT4.9832.3170.0137
ENSRNOG00000016011Plekhg14.9712.3140.0315
ENSRNOG00000037909Ppm1f4.9642.3120.0269
ENSRNOG00000016419Pdlim54.9622.3110.0248
ENSRNOG00000023280Als24.9522.3080.0166
ENSRNOG00000005417Zhx24.9482.3070.0430
ENSRNOG00000017671Rasa34.9442.3060.0403
ENSRNOG00000016848Fzd44.9422.3050.0255
ENSRNOG00000003508LOC1003644004.9422.3050.0244
ENSRNOG00000012394Bcl2l134.9312.3020.0466
ENSRNOG00000018400D4AEL2_RAT4.9312.3020.0303
ENSRNOG00000013707Spata134.9302.3020.0445
ENSRNOG00000002039LOC1003600664.9302.3010.0436
ENSRNOG00000004563Sec24a4.9172.2980.0191
ENSRNOG00000020386D3ZKH4_RAT4.9062.2950.0098
ENSRNOG00000007419Pank34.9002.2930.0128
ENSRNOG00000024533Aer614.8892.2900.0382
ENSRNOG00000027151Lrrc584.8862.2890.0393
ENSRNOG00000030124Ptpn114.8692.2840.0160
ENSRNOG00000006131Mettl24.8462.2770.0271
ENSRNOG00000000407Dcbld14.8342.2730.0412
ENSRNOG00000008061Nuak14.8262.2710.0360
ENSRNOG00000037514Qser14.8212.2690.0136
ENSRNOG00000004959Actr24.8072.2650.0327
ENSRNOG00000028582F1M163_RAT4.7952.2610.0045
ENSRNOG00000043037LOC1003660234.7882.2590.0349
ENSRNOG00000012135F1M2H7_RAT4.7632.2520.0406
ENSRNOG00000031069D4A9A7_RAT4.7492.2470.0462
ENSRNOG00000023109F1LVL2_RAT4.7362.2440.0482
ENSRNOG00000004442RGD13117564.7292.2410.0456
ENSRNOG00000021318Epas14.7232.2400.0138
ENSRNOG00000018099Itch4.7022.2330.0383
ENSRNOG00000038892LOC6861234.6912.2300.0268
ENSRNOG00000000296Aqp64.6852.2280.0310
ENSRNOG00000014901Uggt14.6842.2280.0168
ENSRNOG00000019659Aspa4.6802.2270.0055
ENSRNOG00000010450D4ADY9_RAT4.6622.2210.0220
ENSRNOG000000110666-Mar4.6582.2200.0264
ENSRNOG00000013121Mier34.6472.2160.0408
ENSRNOG00000030894Slco1a64.6402.2140.0068
ENSRNOG00000004964Erbb34.6092.2050.0351
ENSRNOG00000014135Rab11fip44.6072.2040.0453
ENSRNOG00000005052Slc39a94.5942.2000.0454
ENSRNOG00000005276Csnk2a14.5892.1980.0259
ENSRNOG00000015007RGD15655914.5832.1960.0462
ENSRNOG00000002099Wdfy34.5792.1950.0217
ENSRNOG00000001747Pak24.5722.1930.0178
ENSRNOG00000018226Zcchc144.5652.1900.0441
ENSRNOG00000010702Ube3c4.5642.1900.0154
ENSRNOG00000010610Hpgd4.5562.1880.0125
ENSRNOG00000001756D3ZDR3_RAT4.5512.1860.0486
ENSRNOG00000006335Klhl94.5502.1860.0083
ENSRNOG00000016715Kif114.5472.1850.0159
ENSRNOG00000021916Slc16a124.5412.1830.0224
ENSRNOG00000011250Inmt4.5062.1720.0125
ENSRNOG00000013140Pdzd24.5022.1710.0305
ENSRNOG00000012440Msra4.5012.1700.0308
ENSRNOG00000019932Ip6k14.5002.1700.0307
ENSRNOG00000037227Yes14.4992.1700.0412
ENSRNOG00000012054Zmpste244.4982.1690.0179
ENSRNOG00000007370Rnf144a4.4932.1680.0443
ENSRNOG00000022968F1M4Y9_RAT4.4912.1670.0400
ENSRNOG00000011340D3ZMJ4_RAT4.4882.1660.0143
ENSRNOG00000021705D3ZXN6_RAT4.4862.1650.0229
ENSRNOG00000003865Tmigd14.4832.1640.0072
ENSRNOG00000012105F1MAE3_RAT4.4782.1630.0346
ENSRNOG00000011312F1LQ39_RAT4.4752.1620.0366
ENSRNOG00000000127F1LT58_RAT4.4632.1580.0484
ENSRNOG00000022929MTMRC_RAT4.4382.1500.0307
ENSRNOG00000033372Klhl244.4312.1480.0197
ENSRNOG00000008332Smo4.4202.1440.0209
ENSRNOG00000028616Pck14.4182.1430.0219
ENSRNOG00000013281Mib14.4152.1420.0306
ENSRNOG00000011448Eri14.4102.1410.0414
ENSRNOG00000028422Rmnd5a4.4092.1410.0212
ENSRNOG00000014859Rnf1524.4042.1390.0298
ENSRNOG00000001893LOC1003624534.3972.1370.0349
ENSRNOG00000018123Ccny4.3962.1360.0173
ENSRNOG00000016337Slc22a14.3942.1350.0356
ENSRNOG00000003709Kmo4.3892.1340.0166
ENSRNOG00000019939CCND2_RAT4.3862.1330.0383
ENSRNOG000000299474.3772.1300.0399
ENSRNOG00000008346Itgb64.3722.1280.0245
ENSRNOG00000008678Antxr14.3572.1230.0237
ENSRNOG00000029924Klk1l4.3442.1190.0267
ENSRNOG00000043406LOC1003608004.3412.1180.0323
ENSRNOG00000012343Pdp24.3242.1120.0419
ENSRNOG00000009899D3ZWL1_RAT4.3062.1060.0427
ENSRNOG00000003434Trove24.3012.1050.0368
ENSRNOG00000015519Ces1d4.2942.1020.0253
ENSRNOG00000017439Cgnl14.2942.1020.0236
ENSRNOG00000014700Ttc364.2872.1000.0266
ENSRNOG00000007944Edem14.2812.0980.0367
ENSRNOG00000031263Haao4.2462.0860.0200
ENSRNOG00000001647Ets24.2452.0860.0357
ENSRNOG00000008652RGD15649644.2262.0790.0153
ENSRNOG00000023202Usp154.2172.0760.0230
ENSRNOG00000016289Bmpr1b4.2122.0750.0370
ENSRNOG00000015024E9PT54_RAT4.2082.0730.0252
ENSRNOG00000000555Eif4ebp24.1992.0700.0381
ENSRNOG00000008620Smad34.1982.0700.0440
ENSRNOG00000008619Agtrap4.1982.0700.0217
ENSRNOG00000009711Hepacam24.1962.0690.0409
ENSRNOG00000015734Ube3a4.1932.0680.0225
ENSRNOG00000015634SMAD4_RAT4.1892.0670.0277
ENSRNOG00000042519RGD13120264.1822.0640.0380
ENSRNOG00000007564Evc4.1602.0570.0289
ENSRNOG00000008372Vamp74.1602.0570.0433
ENSRNOG00000024671D4AA13_RAT4.1572.0560.0120
ENSRNOG00000004622Calcrl4.1422.0500.0131
ENSRNOG00000009660Enpp64.1402.0500.0247
ENSRNOG00000014750D3ZXU7_RAT4.1382.0490.0176
ENSRNOG00000008694Miox4.1342.0480.0226
ENSRNOG00000004831Arid24.1342.0470.0317
ENSRNOG00000043167Itga94.1242.0440.0349
ENSRNOG00000001770Ehhadh4.1142.0400.0104
ENSRNOG00000042160Tmem167b4.1122.0400.0466
ENSRNOG00000018668Glg14.0952.0340.0172
ENSRNOG00000007985D4ABH6_RAT4.0842.0300.0231
ENSRNOG00000014623F1M3F2_RAT4.0712.0260.0332
ENSRNOG00000002227Kit4.0562.0200.0429
ENSRNOG00000016219Vnn14.0522.0190.0115
ENSRNOG00000008322E9PTI4_RAT4.0352.0130.0418
ENSRNOG00000011358Hipk34.0342.0120.0372
ENSRNOG00000028335Fat44.0172.0060.0190
ENSRNOG00000025554Zfp4454.0092.0030.0365
ENSRNOG00000003388Cenpf3.9901.9960.0146
ENSRNOG00000000614Bicc13.9871.9950.0162
ENSRNOG00000039091D3ZRC4_RAT3.9751.9910.0145
ENSRNOG00000030154Cyp4a23.9621.9860.0422
ENSRNOG000000331723.9521.9830.0176
ENSRNOG00000017466Kif5b3.9491.9810.0128
ENSRNOG00000042879D4A3X0_RAT3.9431.9790.0454
ENSRNOG00000002146Pkd23.9421.9790.0358
ENSRNOG00000012940Vps413.9371.9770.0280
ENSRNOG00000017291Sord3.9281.9740.0133
ENSRNOG00000001606Adamts53.9231.9720.0420
ENSRNOG00000016534D3ZKX0_RAT3.9041.9650.0295
ENSRNOG00000007202Sema3d3.8981.9630.0254
ENSRNOG00000012436Adh63.8971.9620.0137
ENSRNOG00000016334Rod13.8671.9510.0167
ENSRNOG00000018011RGD15644563.8671.9510.0417
ENSRNOG00000039494D4A608_RAT3.8531.9460.0328
ENSRNOG00000014976Acsm23.8501.9450.0330
ENSRNOG00000006636Otud6b3.8171.9320.0439
ENSRNOG00000015849Sepp13.8121.9300.0292
ENSRNOG00000004689Ptdss13.8111.9300.0342
ENSRNOG00000013808Ces2g3.8031.9270.0345
ENSRNOG00000014673Eri23.7911.9220.0429
ENSRNOG00000009819Vezf13.7841.9200.0450
ENSRNOG00000016758Loxl23.7831.9190.0310
ENSRNOG00000010061Gmfb3.7631.9120.0466
ENSRNOG00000023021Msl23.7461.9060.0486
ENSRNOG00000039571Glod53.7421.9040.0370
ENSRNOG00000017600Ptpn93.7391.9030.0329
ENSRNOG00000000590Naglt13.7181.8950.0365
ENSRNOG00000011511Stk243.7161.8940.0398
ENSRNOG00000018279Sfxn13.7121.8920.0213
ENSRNOG00000003953RB3GP_RAT3.7091.8910.0486
ENSRNOG00000024632Atf63.6991.8870.0421
ENSRNOG00000016779Fam120a3.6791.8790.0257
ENSRNOG00000010379Cugbp13.6701.8760.0295
ENSRNOG00000010780Dlc13.6641.8740.0495
ENSRNOG00000003948Llgl13.6591.8710.0486
ENSRNOG00000016183Ipp3.6471.8670.0466
ENSRNOG00000017964Slc22a253.5671.8350.0201
ENSRNOG00000039745Pm20d13.5571.8310.0300
ENSRNOG00000010107PALLD_RAT3.5281.8190.0467
ENSRNOG00000019444D4ADJ6_RAT3.5151.8140.0265
ENSRNOG00000011260Cmbl3.5131.8130.0221
ENSRNOG00000013322DPOLA_RAT3.5051.8100.0498
ENSRNOG00000039278Mcart13.4771.7980.0345
ENSRNOG00000021108Slc22a123.4491.7860.0263
ENSRNOG00000010887RGD13095343.4351.7810.0382
ENSRNOG00000008331RGD13099953.3941.7630.0425
ENSRNOG00000007949Rgn3.3551.7460.0276
ENSRNOG00000011987Cd2ap3.3431.7410.0306
ENSRNOG00000042175B6VQA7_RAT3.3311.7360.0384
ENSRNOG00000012190Cldn23.3241.7330.0347
ENSRNOG00000023972F1M6Q3_RAT3.3231.7330.0322
ENSRNOG00000011763Serp13.3191.7310.0316
ENSRNOG00000004496Rock23.3181.7300.0337
ENSRNOG00000004677Zeb23.3061.7250.0306
ENSRNOG00000013409Gclm3.3011.7230.0338
ENSRNOG00000004302Pah3.2701.7090.0368
ENSRNOG00000010947MMP14_RAT3.2531.7020.0337
ENSRNOG00000011058Utrn3.2471.6990.0378
ENSRNOG00000018215Slc22a63.2461.6980.0377
ENSRNOG00000016456Il333.2341.6930.0319
ENSRNOG00000002541Pds5a3.1641.6620.0449
ENSRNOG00000002680Lamc13.1391.6500.0366
ENSRNOG00000011124Eif4g2-ps13.1251.6440.0380
ENSRNOG00000004009Xpnpep23.1181.6410.0414
ENSRNOG00000010768Kpna43.1141.6390.0499
ENSRNOG00000042249F1LTA7_RAT3.1011.6330.0422
ENSRNOG00000014166Smoc23.0781.6220.0413
ENSRNOG00000002305Slc15a23.0611.6140.0400
ENSRNOG00000005130Ogdh3.0491.6080.0465
ENSRNOG00000018086Slc22a83.0481.6080.0418
ENSRNOG00000010814Bmpr1a3.0061.5880.0432
ENSRNOG00000032885CYC_RAT2.9361.5540.0478

Down-regulated Genes: 15
ENSRNOG00000032087F1LWC2_RAT0.301−1.7340.0328
ENSRNOG000000326090.300−1.7380.0394
ENSRNOG00000033748F1LWC2_RAT0.299−1.7410.0381
ENSRNOG00000025670Shisa30.295−1.7590.0282
ENSRNOG000000291150.284−1.8150.0315
ENSRNOG00000011821S100a40.228−2.1340.0086
ENSRNOG00000007632Zmynd170.211−2.2430.0365
ENSRNOG00000025408D3ZTT0_RAT0.189−2.4030.0422
ENSRNOG00000028844Slc9a50.181−2.4660.0282
ENSRNOG00000006889Ambp0.150−2.7410.0177
ENSRNOG00000028730D3ZI71_RAT0.148−2.7570.0450
ENSRNOG00000026067Wfdc100.144−2.7910.0123
ENSRNOG00000037374D3ZPQ1_RAT0.140−2.8400.0087
ENSRNOG00000033517LOC1003607910.129−2.9500.0009
ENSRNOG00000042909F1LZX4_RAT0.107−3.2290.0466
ENSRNOG00000014578Fxyd40.096−3.3740.0001

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

  1. 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. View at: Publisher Site | Google Scholar
  2. 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. View at: Google Scholar
  3. 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. View at: Publisher Site | Google Scholar
  4. 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. View at: Publisher Site | Google Scholar
  5. 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. View at: Publisher Site | Google Scholar
  6. P. A. Dennery, “Oxidative stress in development: nature or nurture?” Free Radical Biology and Medicine, vol. 49, no. 7, pp. 1147–1151, 2010. View at: Publisher Site | Google Scholar
  7. 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. View at: Publisher Site | Google Scholar
  8. 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. View at: Publisher Site | Google Scholar
  9. 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. View at: Publisher Site | Google Scholar
  10. 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. View at: Publisher Site | Google Scholar
  11. 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. View at: Google Scholar
  12. 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. View at: Publisher Site | Google Scholar
  13. 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. View at: Publisher Site | Google Scholar
  14. 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. View at: Publisher Site | Google Scholar
  15. 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. View at: Publisher Site | Google Scholar
  16. 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. View at: Publisher Site | Google Scholar
  17. 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. View at: Publisher Site | Google Scholar
  18. Y.-L. Tain and C. Baylis, “Determination of dimethylarginine dimethylaminohydrolase activity in the kidney,” Kidney International, vol. 72, no. 7, pp. 886–889, 2007. View at: Publisher Site | Google Scholar
  19. 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. View at: Publisher Site | Google Scholar
  20. 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. View at: Publisher Site | Google Scholar
  21. 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. View at: Publisher Site | Google Scholar
  22. 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. View at: Publisher Site | Google Scholar
  23. 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. View at: Publisher Site | Google Scholar
  24. 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. View at: Publisher Site | Google Scholar
  25. 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. View at: Publisher Site | Google Scholar
  26. 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. View at: Publisher Site | Google Scholar
  27. 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. View at: Publisher Site | Google Scholar
  28. 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. View at: Publisher Site | Google Scholar

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.

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