Sage Journals: Discover world-class research

Abstract

Objective:

The current study considers changes of the postnatal brainstem cell number and angiotensin receptors by maternal protein restriction (LP) and LP taurine supplementation (LPT), and its impact on arterial hypertension development in adult life.

Methods and results:

The brain tissue studies were performed by immunoblotting, immunohistochemistry, and isotropic fractionator analysis. The current study shows that elevated blood pressure associated with decreased fractional urinary sodium excretion (FE_Na) in adult LP offspring was reverted by diet taurine supplementation. Also, that 12-day-old LP pups present a reduction of 21% of brainstem neuron counts, and, immunohistochemistry demonstrates a decreased expression of type 1 angiotensin II receptors (AT₁R) in the entire medial solitary tract nuclei (nTS) of 16-week-old LP rats compared to age-matched NP and LPT offspring. Conversely, the immunostained type 2 AngII (AT₂R) receptors in 16-week-old LP nTS were unchanged.

Conclusion:

The present investigation shows a decreased FE_Na that occurs despite unchanged creatinine clearance. It is plausible to hypothesize an association of decreased postnatal nTS cell number, AT₁R/AT₂R ratio and FE_Na with the higher blood pressure levels found in taurine-deficient progeny (LP) compared with age-matched NP and LPT offspring.

Keywords

Arterial hypertension low-protein diet taurine angiotensin receptors solitary tract nucleus kidney function natriuresis

Introduction

The concept of fetal programming suggests that the fetus is programmed in uteri to develop a number of adult diseases, including arterial hypertension and diabetes mellitus.¹ Intrauterine growth restriction (IUGR) has been associated with maternal low protein intake and, although the specific nature of this insult is unclear, a number of mechanisms have been proposed. In uteri programming of hypertension via alteration of the renin angiotensin system (RAS) before birth has attracted great attention. Since these initial discoveries, most intrinsic components of the RAS, including angiotensinogen, angiotensin, and converting enzymes, have been well described and demonstrated in different areas of the central nervous system (CNS).^2,3 Notably, most previous studies in this ﬁeld have focused on the central and peripheral angiotensin receptors and their roles in prenatal imprinting. The challenge for all of us is to understand in depth the mechanisms by which antenatal stress may alter normal neurophysiology of the brain RAS. Intrauterine malnutrition has been known to induce alterations in the development of several tissues, among which are the CNS.³ The role of the CNS in the control of blood pressure and hydrosaline homeostasis has been demonstrated by several studies.^4,5 Also, the central role of the RAS in the control of blood pressure and hydroelectrolyte homeostasis has been widely demonstrated.^6,7 The medial solitary tract nucleus (nTS), the central site of termination of baroreceptive afferents, is intimately involved in arterial pressure control. This nucleus contains a high density of angiotensin II (AngII) AT₁ receptors (AT₁R) located both presynaptically, on vagal and carotid sinus afferents, and on interneurons.⁸ However, in the IUGR models the cytology pattern of medial solitary tract nuclei (nTS) modulation of AngII receptors is not well known. AngII lowers blood pressure and heart rate after injection of low doses in the nTS, as previously reported in several strains of rats.^9,10 This response can be blocked completely by the nTS injection of an AT₁R antagonist.¹¹ The current study pays attention to changes of the postnatal nTS angiotensin receptors by maternal protein restriction (LP), and its impact on in uteri programming of hypertension in adult life. Taking in account the above findings, the purpose of the present study, firstly, was to determine whether maternal protein restriction during whole pregnancy alters the nTS cytological pattern and expression of AT₁R and AT₂R in 16-week-old (LP) offspring; these data were compared with those of age-matched appropriate normal-protein ingestion (NP) controls. On the other hand, a study has also shown previously that the level of taurine (2β-amino ethanesulphonic acid) is markedly reduced in the plasma of fetuses of dams fed a low-protein diet.¹² Maternal taurine supplementation of the LP diet restored to normal the taurine levels in fetal plasma.¹³ Thus, the second aim of this study was to determine if taurine, added to LP content chow (LPT) of these dams, could provide prevention against hypertension development when compared to LP offspring. We also hypothesized that arterial hypertension in adult life may result, at least in part, from nTS disorders in association with modified urinary sodium handling, evaluated by lithium clearance, in conscious maternal LP intake rats, when compared with their appropriate experimental controls (NP and LPT groups).

Materials and methods

Animals

The experiments were conducted on age-matched, female offspring of sibling-mated Wistar rats (0.250–0.300 kg) allowed free access to water and normal rat chow. The general guidelines established by the Brazilian College of Animal Experimentation (Protocol #2575-1) were followed throughout the investigation. Our local colonies originated from a breeding stock supplied by the University of Campinas Animal Breeding Center, Campinas, SP, Brazil. Immediately after weaning at 3 weeks of age, animals were maintained under controlled temperature (25^oC) and lighting conditions (0700 h–1900 h), with free access to tap water and standard rodent laboratory chow (Nuvital, Curitiba, PR, Brazil with Na⁺ content: 135 ± 3 µEq/g; K⁺ content: 293 ± 5 µEq/g) and followed up to 12 weeks of age. Animals were mated and the day that sperm were seen in the vaginal smear was designated as day one of pregnancy. The dams were maintained on isocaloric standard rodent laboratory (with normal protein content (NP), 17% protein), low protein content (LP) (6% protein) or 2.5% taurine added to the LP content chow (LPT) ad libitum intake throughout the entire pregnancy. The maternal body weight gain for all groups was determined between the first and third gestational period. All groups resumed the NP chow intake after delivery. The male pups were followed and maintained with normal chow until adulthood. The offspring food consumption (subsequently normalized for body weight) and body weight were determined every day, all over 16 weeks. The data relating to body weight were obtained on a daily basis. However, in this study data were presented as the weekly average of all daily weights.

Blood pressure measurement

Systolic blood pressure was measured in conscious 6-, 8-, 10-, 12-, 14- and 16-week-old rats (LP, LPT and NP; n = 12 for each group) by an indirect tail-cuff method using an electrosphygmomanometer (IITC Life Science – BpMonWin Monitor Version 1.33) combined with a pneumatic pulse transducer/amplifier. This indirect approach allowed repeated measurements with a close correlation (correlation coefficient = 0.975), compared to direct intra-arterial recording.^14–17 The mean of three consecutive readings represented the blood pressure.

Total cell and neuron quantification of the medulla oblongata

The cell and neuron quantification followed the technique described by Herculano-Houzel and Lent (2005). Briefly, five 12-day-old and adult offspring from different mothers of NP, LP, and LPT rats were sacrificed and perfused transcardially with saline, followed by 4% buffered paraformaldehyde. The medulla oblongata was removed from the brain using the foramen magnum as the inferior limit of anatomical landmarks for dissection. A suspension of nuclei is obtained through mechanical dissociation of the fixed brain tissues in a standard solution (40 mM sodium citrate and 1% Triton X-100), using a 40-ml glass Tenbroeck tissue homogenizer. Using at least 1 ml of dissociation solution per 100 mg of brain tissue and grinding until the smallest visible fragments are dissolved achieves complete homogenization. The homogenate is collected with a Pasteur pipette and transferred to 15-ml centrifuge tubes. The grinding pestle and tube are washed several times with dissociation solution and centrifuged for 10 min at 4000 g. Pelleted nuclei are then suspended in phosphate-buffered saline (PBS) containing 1% 4’,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR, USA), to make all of the nuclei visible under ultraviolet illumination. After sufficient agitation, 5-µl aliquots are removed for determination of nuclei density in a hemocytometer. DAPI-stained nuclei are counted under a fluorescence microscope at 400× magnification. Once nuclear density in the suspension is determined by averaging over at least eight samples, the total number of cells in the original tissue is estimated by multiplying mean nuclear density by total suspension volume. For estimates of total neuron number, a 200–500-µl aliquot is removed from the nuclear suspension and immunoreacted for NeuN. Nuclei in the aliquot are collected by centrifugation, resuspended in a 0.2-M solution of boric acid, pH 9.0, and heated for one hour at 75°C for epitope retrieval. Subsequently, nuclei are again collected by centrifugation, washed in PBS, and incubated overnight at room temperature with anti-NeuN mouse immunoglobulin G (IgG) (1:300 in PBS; Chemicon, Temecula, CA, USA). After being washed in PBS, nuclei are incubated in cyanine 3-conjugated goat anti-mouse IgG secondary antibody (1:400 in 40% PBS, 10% goat serum, and 50% DAPI; Accurate Chemicals, Westbury, NY, USA) for two hours, collected by centrifugation, washed in PBS, and then suspended in a small volume of PBS for counting under the fluorescence microscope. Total number of nonneuronal nuclei is calculated by subtracting the number of NeuN containing nuclei from the total number of nuclei.¹⁸

Renal function evaluation

The renal function tests were performed at 8 and 16 weeks of age in unanesthetized, unrestrained NP (n = six), LP (n = six) and LPT (n = six) male rats. Briefly, 14 hours before the renal test, 60 µmol LiCl 100 g⁻¹ body weight was given by gavage. After an overnight fast, each animal received a load of tap water by gavage (5% of the body weight), followed by a second load of the same volume, one hour later, and spontaneously voided urine was collected over a 120-minute period into a graduated centrifuge tube. At the end of the experiment, blood samples were drawn through the cardiac puncture in anesthetized rats and urine and plasma samples were collected for analysis.^14–17

Western blot

Brain tissue extraction and immunoblotting was performed as previously described.^18,19 Briefly, NP, LP and LPT (n = five for each group) rats (16 weeks old), were anesthetized and subjected to craniotomy. Brainstems were obtained and homogenized in freshly prepared ice-cold buffer (1% Triton X-100, 100 mM Tris, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.01 mg aprotinin/ml). Insoluble material was removed by centrifugation (10,000 g) for 25 minutes at 4^oC. Protein quantification was performed using the Bradford method. For immunoblotting of total protein extracts, 0.2 mg total protein were suspended in Laemmli sample buffer, boiled for 5 minutes and loaded onto the electrophoresis gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and blot followed the same steps as described above for immunoblotting. The nitrocellulose transfers were probed with specific antibodies (AT1_R 1:1000, AT2 _R 1:1000). The blots were subsequently incubated in peroxidase-conjugated secondary antibodies (1:10,000). Immunoreactive bands were detected using the enhanced chemiluminescence method (RPN 2108 ECL Western Blotting analysis system; Amersham Biosciences) and detected by pre-flashed Kodak XAR film. Band intensities were quantified by optical densitometry of developed chemiluminescence (Scion Image software, ScionCorp, Frederick, MD, USA). To ensure equal loading, membranes were stained with Coomassie Brilliant Blue dye before blotting. As shown in Figure 1, all membranes were also incubated with β-actin antibody to discard possible inequalities in protein loading and/or transfer. Only homogeneously stained membranes were employed in the study.

Figure 1.

Creatinine clearance (C_Cr), fractional sodium excretion (FE_Na), proximal (FEP_Na) and post-proximal (FEPP_Na) fractional sodium excretion and fractional potassium excretion (FE_K) in control (NP), maternal protein-restricted (LP) and, LP taurine-supplemented 8-week- (Panel A) and 16-week-old (Panel B) offspring. See Results for statistical analysis details. The data are reported as the means ± SD. *p ≤ 0.05 vs. NP (analysis of variance (ANOVA); post-hoc Bonferroni’s test).

Tissue processing, histology and immunohistochemical procedures

Sixteen-week-old male rats from the NP (n = five), LP (n = five), and LPT (n = five) groups were used. The rats were anesthetized with a mixture of ketamine (75 mg.kg⁻¹ body weight, intraperitoneally (i.p.)) and xylasine (10 mg.kg⁻¹ body weight, i.p.) and monitoring the corneal reflex controlled the level of anesthesia. The animals were then perfused with saline containing heparin (5%) for 15 minutes under constant pressure, followed by perfusion with 0.1 M phosphate buffer (PB; pH 7.4) containing 4% (w/v) paraformaldehyde and 0.1 M sucrose for 25 minutes. After the perfusion, brains were removed and placed in the same fixative for two hours for paraffin embedding. For immunohistochemical analysis we used anti-AT₁R, AT₂R antibodies (Santa Cruz Biotech Inc, CA, USA). Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0) boiling in a microwave oven (1300 watts) twice for five minutes each. Proteins were immunohistochemically detected using the avidin-biotin-peroxidase method. Briefly, deparaffinized 5-µm-thick sections on poly-l-lysine coated slides were treated with 3% H₂O₂ in PBS for 15 minutes, nonfat milk for 60 minutes, primary antibodies for 60 minutes, and avidin-biotin-peroxidase solution (Vector Laboratories Inc, CA, USA, 1:1:50 dilution). Chromogenic color was accomplished with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich Co., St. Louis, MO, USA) as the substrate to demonstrate the sites of peroxidase binding. The slides were counterstained with Harris’s hematoxylin.

Antibodies and chemicals

SDS-PAGE and immunoblotting reagents were obtained from Bio-Rad (Richmond, CA, USA). Hepes, PMSF, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, AngII, and bovine serum albumin (BSA) (fraction V) were from Sigma Chemical Co. (St Louis, MO, USA) and nitrocellulose membranes were from Amersham Corp. (Aylesbury, Bucks, UK). Antibodies against AT₁R, AT₂R, (rabbit polyclonal, AB-1565) for immunoblotting was from Millipore and Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Secondary antibodies and conjugated complexes utilized in immunohistochemistry were from Vector Laboratories Inc (Burlingame, CA, USA). Sodium pentobarbital was from Cristália (São Paulo, Brazil).

Data presentation and statistical analysis

All numerical results are expressed as the mean ± SD of the indicated number of experiments. Plasma and urine sodium, potassium, and lithium concentration were measured by flame photometry (Micronal, B262, São Paulo, Brazil), while the creatinine concentrations and plasma osmolality were determined spectrophotometrically (Instruments Laboratory, Genesys V, USA) and by Wide-range Osmometer (Advanced Inst. Inc, MA, USA), respectively. Creatinine clearance was used to estimate glomerular filtration rate (GFR) and lithium clearance (C_Li) was used to assess proximal tubule output.^14–17 Fractional sodium excretion (FE_Na) was calculated as C_Na/C_Cr × 100, where C_Na is sodium clearance and C_Cr is creatinine clearance. The fractional proximal (FEP_Na) and post-proximal (FEPP_Na) sodium excretion was calculated as C_Li/C_Cr × 100 and C_Na/C_Li × 100, respectively. Data obtained over time were analyzed using one-way analysis of variance (ANOVA). Post hoc comparisons between selected means were performed with Bonferroni’s contrast test when initial ANOVA indicated statistical differences between experimental groups. Comparisons involving only two means within or between groups were carried out using a Student’s t test. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using the Scion Image software (ScionCorp). The level of significance was set at p ≤ 0.05.

Results

There were no significant differences between plasma sodium, potassium, lithium, osmolality and urine flow (Table 1) in NP rats, compared with the LP group. The maternal body weight for all groups, at first (NP1: 207.8 ± 11.23 g; LP1: 216.2 ± 16.7 g and LPT1: 203.9 ± 15.03 g) and third gestational week (NP3: 265.8 ± 19.42 g; LP3: 255.2 ± 21.82 g and LPT3: 251.2 ± 16.6 g), was similar (p > 0.05 for each group). Otherwise, the maternal gestational body weight gain was significantly higher in NP and LPT (NP1 vs. NP3: 59%; LPT1 vs. LPT3: 48%,p < 0.0001) when compared to LP weight gain (LP1 vs. LP3: 32.75%, p < 0.01). The data relating to offspring body weights were obtained on a daily basis. However, in this study data were presented as the weekly average of all daily weights. In general, food intake and therefore sodium intake were similar, when normalized by body weight, in male offspring of NP, LP, and LPT groups during the investigation. The LP male pup body weight was significantly reduced when compared to that of NP and LPT pups (5.98 ± 0.6 g vs. 6.65 ± 0.46 g and 5.98 ± 0.6 g vs. 6.58 ± 0.32 g, respectively, p = 0.005). However, the body masses at 16 weeks old (p > 0.05) were similar to those observed in NP and LPT age-matched groups (Table 1). The arterial blood pressure increased significantly more in LP than in NP rats from 6 to 16 weeks of age; LP pressure increased from 116.2 ± 6.5 mmHg to 137.9 ± 6.9 mmHg as compared with a smaller and nonsignificant rise from 114.7 ± 7.4 mmHg to 118.8 ± 8.7 mmHg and from 114.0 ± 7.4 mmHg to 123.2 ± 5.6 mmHg (n = six for each group; p = 0.01 vs. NP) in NP and LPT, respectively. In LP, the significant rise in the arterial pressure appeared after 12 weeks of age (Figure 2).

Table 1.

Serum sodium, potassium, lithium levels, osmolality, urine flow, and body weight (b.w.) as related to age and blood pressure (BP) in maternal protein-restricted (LP), taurine-supplemented (LPT) and maternal normal-protein intake offspring (NP).

Groups	Na⁺(mM)	K⁺(mM)	Li⁺(µM)	Osmolality(mOsm.kg⁻¹H₂O)	V(µl/min/100 g b.w.)	Body weight (g)16 weeks old	BP (mmHg)16 weeks old
NP (n = 6)	136 ± 2.8	4.6 ± 0.3	84 ± 21	293 ± 8	51.2 ± 12.5	381 ± 25.5	118.8 ± 8.7
LP (n = 6)	141 ± 4.3	4.2 ± 0.5	81 ± 19	297 ± 7	51.2 ± 6.8	415.4 ± 16	137.9 ± 6.9^a
LPT (n = 6)	139 ± 3.7	4.3 ± 0.6	83 ± 18	296 ± 9	50.4 ± 7.6	401.8 ± 22.8	123.2 ± 5.6

Data are reported as means ± SD. ^ap ≤ 0.05 vs. NP (one-way analysis of variance (ANOVA); post-hoc Bonferroni’s test).

Figure 2.

Effect of age on brainstem weight (per 100 g body weight, 3(a), on top); brainstem total cellular quantification (per mg of the brainstem mass, 3(b), on top) and, brainstem neurons quantification (per mg of the brainstem mass, 3(c), on bottom) in male offspring of protein-restricted, taurine-supplemented and standard-diet mothers during gestation. See Results for statistical analysis details. The data are reported as the means ± SD. In all experiments, n = 6; *p ≤ 0.05 vs. NP (analysis of variance (ANOVA); post hoc Bonferroni’s test).

Total cell and neuron quantification of the medulla oblongata

The LP male pup (12 days of age) brainstem weight, normalized by body weight for each experimental group, was significantly reduced when compared to that of NP (less about 15%) and LPT (less about 29%) pups (311 ± 31.7 mg% vs. 358.9 ± 41.9 mg% and 311 ± 31.7 mg% vs. 404 ± 34.1 mg%; p = 0.03), respectively. However, the brainstem masses at 16 weeks old (p > 0.05) per 100 g of body weight were similar to those observed in NP and LPT age-matched offspring (Figure 3(a), on top). We find a similar total number of cells per brainstem weight (in mg) in 12-day and 16-week of age-matched NP adult offspring (12-day: 33.5 ± 7.3 × 10⁴; 16-week: 13.5 ± 2.5 × 10⁴ cells; n =five) when compared to medulla oblongata of the LP (12-day: 27.4 ± 73.5 × 10⁴; 16-week: 11.73 ± 1.4 × 10⁴ cells; n = 5) and LPT (12-day: 31.7 ± 5.1 × 10⁴; 16-week: 9.48 ± 3.21 × 10⁴ cells; n = five) (Figure 3(b), on top). However, the brainstem total cells for 12-day- and 16-week-old (p > 0.05) per mg de medulla oblongata were similar to those observed in NP and LPT age-matched offspring, there is a significant difference between 12-day and 16-week-old groups (Figure 3(a), on top). Therefore, the pup (12-day-old) rat contains almost three times as many cells per mg of the brainstem (averaging 30.42 ± 2.01 × 10⁴ cells) as the brainstem of 16-week-old offspring (averaging 10.62 ± 1.2 × 10⁴ cells, p < 0.05). Conversely, the neuron quantification per brainstem weight (in mg) shows an expressive reduction of neurons (about 21%) in 12-day-old offspring (40 ± 5.55 × 10³ neurons, p < 0.05) when compared to NP (50.8 ± 8.43 × 10³ neurons) and LPT (52.5 ± 7.03 × 10³ neurons) offspring (Figure 3(c), on bottom). Otherwise, no difference was observed among LP, NP and LPT groups at 16-week-old rates. Therefore, the pup (12-day-old) offspring contain almost two and half times as many cells per mg of the brainstem (averaging 47.7 ± 7.0 × 10³ neurons) than in the brainstem of 16-week-old offspring (averaging 13.9 ± 1.04 × 10³ neurons, p < 0.05).

Figure 3.

Effects of age on arterial pressure in male offspring of protein-restricted, taurine-supplemented and standard-diet mothers during gestation. See results for statistical analysis details. The data are reported as the means ± SD. In all experiments, n = 6; *p ≤ 0.05 vs. NP (analysis of variance (ANOVA); post hoc Bonferroni’s test).

Renal function data

The data for renal function in the 8- and 16-week-old offspring of the NP, LP, and LPT groups are summarized in Figure 3. The urinary flow rates (Table 1) and the GFR, estimated by C_Cr, after oral water load, did not significantly differ among the groups during the renal tubule sodium handling studies. FE_Na was significantly lower in both LP (0.74 ± 0.03%) and LPT (0.79 ± 0.02%) offspring beyond 8 weeks old, when compared with the NP age-matched group (0.95 ± 0.07%; p = 0.0001). However, the FE_Na of 16-week-old offspring remained significantly decreased in the LP, but not in the LPT (0.98 ± 0.12%) group, as follows: LP 16-week: 0.72 ± 0.03% vs. NP 16-week: 1.05 ± 0.1% (p = 0.0001), respectively. The decreased FE_Na in LP offspring was accompanied by a significant decrease in FEP_Na when compared only with the NP age-paired control group (p = 0.01). This decreased FE_Na occurred despite unchanged C_Cr and a significant enhance in FEPP_Na in the 8-week-old groups (LP: 1.08 ± 0.13% vs. NP: 0.84 ± 0.11%, p = 0.006 and vs. LPT: 0.84 ± 0.11%, NS), as well as in 16-week-old offspring (LP: 1.12 ± 0.15% vs. NP: 0.73 ± 0.14%, p = 0.002 and vs. LPT: 0.92 ± 0.16%, NS) (Figure 3). This consistent fall in FE_Na and FEP_Na, and the increase in FEPP_Na, produced by protein-intake restriction during pregnancy, was followed by unaffected kaliuresis in the entire experimental groups of the present investigation.

Western blot analysis of AT1_R and AT2_R expression

Western blot analysis in male offspring of NP, LP, and LPT rat brainstems in 12-day- and 16-week-old offspring yielded a single band at the expected weight of corresponding proteins. The expressions of AT₁R proteins studied in the whole medulla oblongata tissue extracts of 12-day- (NP: 100 ± 6.42%; LP: 52 ± 27.56% and LPT: 61.14 ± 19.1%, p > 0.06) and 16-week-old LP and LPT rats (Figure 4), despite being lower were not statistically significant when compared to those of NP and LPT rats (Figure 4). Additionally, the expression of the AT₂R proteins in the brainstem of 12-day- (NP: 100 ± 36.41%; LP: 81.35 ± 24.05% and LPT: 62.6 ± 14.5%) and 16-week-old LP and LPT offspring (p = 0.489) was unchanged when compared with the age-paired NP group (Figure 4).

Figure 4.

Effects of maternal protein restriction (LP) on expression of entire brainstem AT₁R and AT₂R proteins. This figure shows the results obtained in whole-tissue extracts that were immunoblotted for AT₁ and AT₂ receptors protein content verification in 16-week-old NP, LP, and LPT brainstems. The results of scanning densitometry were expressed as relative to NP, assigning a value of 100% to the control rats. AT₁R: type 1 AngII receptors; AT₂R: type 2 AngII receptors. Columns and bars represent the mean ± SD *p < 0.05 vs. NP (analysis of variance (ANOVA); post hoc Bonferroni’s test).

nTS immunohistochemical analysis of AT₁R and AT₂R

The nTS AT₁R immunoreactivity was markedly decreased in 16-week-old LPs comparatively to the NP age-matched offspring group. Otherwise, in 16-week-old LPTs, the immunoreactivity to AT₁R presented similarly to that observed in NP offspring of the same age and in this similar brainstem nucleus (Figure 5). The unchanged AT₁R blotting in the whole medulla oblongata extracts of 16-week-old LP rats may result in uneven AT₁R and AT₂R expression in several nuclei, as revealed by qualitative immunohistochemistry of different parts of analyzed nTS structures. At the same time, the nTS AT₂R immunoreactivity was markedly decreased in 16-week-old LP and LPT rats when compared with age-matched NP offspring (Figure 6).

Figure 5.

Effects of maternal protein restriction (LP) on AT₁R immunolocalization in 16-week-old rat transversal sections of tract solitary nucleus (nTS) compared to NP and LPT offspring. The immunoreactivity for this receptor was reduced in LP (B and b, small letter in detail), LPT (C and c) when compared to NP (A and a).⁵¹ AT₁R: type 1 angiotensin II receptors; NP: normal protein; LPT: maternal protein restriction with taurine supplementation.

Figure 6.

Effects of maternal protein restriction (LP) on AT₂R immunolocalization in 16-week-old rat transversal sections of tract solitary nucleus (nTS) compared to NP and LPT offspring. The immunoreactivity for this receptor was reduced in LP (B and b, small letter in detail) and LPT (C and c) when compared to NP (A and a).⁵¹ AT₂R: type 2 angiotensin II receptors; NP: normal protein; LPT: maternal protein restriction with taurine supplementation.

Discussion

IUGR is a pregnancy complication associated with adverse outcomes such as neurodevelopmental handicaps.^20,21 The morpho-functional organization of the CNS in mammals is established during the prenatal and early postnatal periods of development through the synthesis of cellular components, neurogenesis, and gliogenesis, migration, and cell differentiation. Although the precise mechanism by which blood pressure rises in LP offspring remains to be elucidated, neural activity and renal control of the fluid and electrolyte balance are thought to play a dominant role in the long-term control of arterial blood pressure. In uteri programming of arterial hypertension via alteration of the RAS before birth has attracted great attention. The present study shows that the maternal body weight gain during the gestational period was significantly higher in NP and LPT when compared to LP weight gain. These maternal findings are associated with a significantly reduced LP male pup body weight compared to that of NP and LPT pups. Our data, confirming a prior report, indicate that LP kidneys even after higher blood pressure development excrete lesser amounts of salt under basal conditions than kidneys of NP rats.^19,22 Also, the present findings confirm previous studies in different areas of CNS,²³ but not in the brainstem, showing that maternal LP restriction during prenatal life decreases the mass and neuronal proliferation (about 21%) in this encephalic area. These disorders of the fetal brain areas, including the brainstem, may affect fetal neural cell maturation and hence have profound consequences in functional neural postnatal life. The nTS, the central site of termination of baroreceptive afferents, is intimately involved in arterial pressure control. This nucleus contains a high density of AT₁Rs located both presynaptically, on vagal and carotid sinus afferents, and on interneurons.²⁴ AngII lowers blood pressure and heart rate after injection of low doses in the nTS, as previously reported in several strains of rats.^9,10 This response was completely blocked by the nTS injection of an AT₁R antagonist.¹¹ The present study has shown a striking reduction of AT₁ receptors in the nTS of the maternal LP offspring when compared with unchanged AT₁R density in NP and taurine-treated LPT rats. The pattern of distribution of AT₁R has been identified on local neurons of the nodose ganglion and nTS as well as fiber terminals that project to these sites by anatomical, molecular or electrophysiological^24–26 techniques. Study has revealed that AngII may act directly on nTS neurons to stimulate efferent pathways responsible for parasympathetic control of blood pressure. However, at least in part, the AngII-mediated hypotensive effect may result from inhibition of sympathetic nervous system activity by direct connections of vagal sensory afferent fibers with cells in the A₂-catecholamine cell group in the ventral nTS.²⁷ We did not completely rule out that the blood pressure fall in LPT may have resulted from receptors on presynaptic afferent-fiber ends in the nTS that are most likely of vagal origin. In this case, studies have shown that substance P (SP) antagonists¹⁰ attenuate the decreased pressure response elicited by AngII in the nTS.^28,29,30 The current study demonstrates a decreased nTS AT₁R expression, by immunohistochemistry, in 16-week-old LP offspring when compared to NP and LPT groups. The nonsignificant decreased AT₁R blotting results, in the whole brainstem extracts of 16-week-old LP rats, may result in uneven angiotensin receptor expression in several subnuclei, as revealed by qualitative immunohistochemistry of rostral, medial and caudal subnuclei analyzed in nTS structures.

Maternal dietary protein restriction during pregnancy is associated with renal morphological and physiological changes. Different mechanisms can contribute to this phenotype: exposure to fetal glucocorticoid, alterations in the components of the RAS, apoptosis, and DNA methylation. An LP diet during gestation decreases the activity of placental 11ß-hydroxysteroid dehydrogenase, exposing the fetus to glucocorticoids and resetting the hypothalamic-pituitary-adrenal axis in the offspring. The abnormal function/expression of AngII receptors during any period of life may be the consequence or cause of renal adaptation. AT₁R is up-regulated, compared with controls, on the first day after birth of offspring born to LP diet mothers, but this protein appears to be down-regulated by 12 days of age and thereafter. In these offspring, AT₂R expression differs from controls at one day of age, but is also down-regulated thereafter, with low nephron numbers at all ages: from the fetal period, at the end of nephron formation, and during adulthood.^19,20 The current investigation also shows an early and pronounced decrease in FE_Na in maternal LP offspring beyond 8 weeks of age when compared to age-matched NP. The decreased FE_Na was accompanied by a fall in FEP_Na and occurred despite unchanged C_Cr and an enhanced FEPP_Na. In this case, fluid is reabsorbed to the same degree, resulting in the concentration in the end of the proximal tubule being the same as in the beginning. In other words, the reabsorption in the proximal tubule is isosmotic without a change in the plasma osmolality. These effects were associated with a significant extracellular isotonic expansion and supposedly enhance arterial blood pressure in the LP group, but the precise mechanism of these phenomena remains unknown. While circulating AngII tends to retain sodium by a direct renal action,⁷ as well as through aldosterone release from the adrenal gland, stimulation of brain AngII receptors has been reported to induce natriuresis.^31,32 The mechanism by which central AngII induces its natriuretic effects remains to be elucidated. Several possibilities may be considered. First, the CNS may directly influence renal sodium excretion through neural routes. Secondly, hemodynamic factors may be responsible for the alterations in electrolyte excretion. Thirdly, natriuresis may result from fluctuations in the level of neural factors that influence tubular sodium handling. There is considerable evidence to support a role for the sympathetic nervous system in the control of urinary sodium excretion.^4,5 Otherwise, some neurons in the nTS that express AT₁R have polysynaptic connections to peripheral organs such as the kidney via renal sympathetic nerves.^33,34 A previous study reported that, in conscious rats, central AngII induces an immediate reduction in the efferent renal nerve and enhanced renin-angiotensin dipsogenic and natriuretic response.^17,35,36 Otherwise, further studies are needed to evaluate the repercussion of taurine supplementation directly on kidney morphology and development, and vascular reactivity.

The second major isoform of the angiotensin receptor, AT₂R, is widely expressed at high levels in fetal tissues, and decreases rapidly after birth.^37,38 Alterations in AT₂R signaling may change the delicate balance between growth stimulation and inhibition, leading to alterations in development. However, according to current and previous studies, there is no strong evidence for AT₂Rs in the medial nTS of the rat that would account for the residual actions of AngII. Also, it is plausible to consider that the decreasing AT₁R/AT₂R ratio associated with attenuated AngII hypotensive and natriuretic responses, mediated by neural pathways with origin in nTS, is implicated with the higher blood pressure levels in adult LP offspring. These findings may also occur by opposite action of AT₂Rs to AT₁Rs in specific brain areas.³⁹

A study has also shown previously that the level of taurine is markedly reduced in the plasma of fetuses of dams fed an LP diet.¹² Additionally, taurine supplementation of the maternal LP intake restored to normal the fetal plasma taurine concentration.¹³ This β-amino acid can be regarded as an essential amino acid during fetal life, because the capacity to synthesize taurine is low or absent in the human fetus.^40,41 Consequently, the fetus is dependent on highly efficient active placental transport for a continuous supply of this amino acid. Apart from this, the physiologic function of this amino acid remains elusive. In animal experiments, including primate models, taurine deficiency during pregnancy and lactation is associated with growth failure, abnormal cerebellar development, neurologic deficits, retinal degeneration, and cardiac damage.⁴² The role of taurine protecting against oxidative damage has been described in a variety of cell types. This amino acid, possibly through its antioxidant activity and regulation of intracellular calcium ﬂux, can prevent the death of endothelial cells.⁴³ Taking into account the above findings, we may suppose that progressive enhanced blood pressure beyond 8 weeks of age in LP offspring may be associated with that pronounced reduction in cellularity and AT₁R density in the nTS. Conversely, this finding reverted by diet taurine-supplementation (in LPT), normalizing the arterial pressure and urinary sodium excretion in adult offspring. Moreover, AngII has been also shown to stimulate Ca²⁺ cellular transport.^44,45

Taurine may also reduce blood pressure through attenuation of peripheral AngII activity, enhancement of the kinin-kallikrein system in the kidney,^46,47 or decreasing levels of epinephrine and norepinephrine. Additionally, as observed in the current study, taurine supplementation effectively normalizes high blood pressure in the most common animal models of hypertension, including: spontaneously hypertensive rats (SHR),⁴⁸ deoxycorticosterone acetate (DOCA)-salt rats,⁴⁹ and Dahl-S rats.⁵⁰

Conclusion

In conclusion, although the precise mechanism responsible for the subsequently enhanced sodium retention response in LP offspring rats is still unclear, the current data suggest that maternal low taurine ingestion may lead to changes in nTS cardiovascular and sympathetic nerve activity that are conducive to excess hydroelectrolytic tubule reabsorption, and that this might potentiate the programming of adult hypertension. This raises the possibility that taurine during the gestational period may inhibit several other actions of AngII through the regulation of an early step in the signaling pathway of AngII. In fact, in the present study it is plausible to suppose an association of decreasing nTS neuronal cellularity, AT₁R/AT₂R ratio, and water-electrolyte renal excretion with the higher blood pressure levels found in LP (taurine-deficient progeny), compared with age-matched NP and LPT offspring. The mechanisms by which the fetal programming causes these disorders remain unknown and further studies are needed in this regard.

Footnotes

Acknowledgements

The authors wish to thank Bs. Ize Penhas de Lima for expert technical assistance.

Conflict of interest

None declared.

Funding

This work was supported by grants from the National Council for Scientific and Technological Development (CNPq) (No.500868/91-3), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the São Paolo Research Foundation (FAPESP) (10/52696-0).

References

Ashton

. Perinatal development and adult blood pressure. Brazilian J Med Biol Res 2000; 33: 731–740.

Saavedra

. Brain angiotensin II: New developments, unanswered questions and therapeutic opportunities. Cell Mol Neurobiol 2005; 25: 485–512.

De Lima

Scabora

Lopes

. Early changes of hypothalamic angiotensin II receptors expression in gestational protein-restricted offspring: Effect on water intake, blood pressure and renal sodium handling. J Renin Angiotensin Aldosterone Syst. Epub ahead of print 30 August 2012.

Gontijo

JAR

Garcia

Figueiredo

. Renal sodium handling after noradrenergic stimulation of the lateral hypothalamus area in rats. Brazilian J Med Biol Res 1992; 25: 937–942.

DiBona

. Nervous kidney. Interaction between renal sympathetic nerves and renin-angiotensin system in the control of renal function. Hypertension 2000; 36: 1083–1088.

Ferguson

Washburn

Latchford

. Hormonal and neurotransmitter roles for angiotensin in the regulation of central autonomic function. Exp Biol Med 2001; 226: 85–96.

Hall

Brands

. Chapter 40: The renin-angiotensin-aldosterone systems. In: Seldin

Giebisch

(eds) The kidney: Physiology and pathophysiology. New York, NY: Lippincott Williams & Wilkins, 2000, pp. 1009–1046.

Healy

Rettig

Nguyen

. Quantitative autoradiography of angiotensin II receptors in the rat solitary-vagal area: Effects of nodose ganglioectomy or sinoaortic denervation. Brain Res 1989; 484: 1–12.

Diz

Barnes

Ferrario

. Hypotensive actions of microinjections of angiotensin II into the dorsal motor nucleus of the vagus. J Hypertens 1984; 2: 53–56.

10.

Diz

Fantz

Benter

. Acute depressor actions of angiotensin II in the nucleus of the solitary tract are mediated by substance P. Am J Physiol 1997; 273: R28–R34.

11.

Fow

Averill

Barnes

. Mechanisms of angiotensin-induced hypotension and bradycardia in the medial solitary tract nucleus. Am J Physiol 1994; 267: H259–H266.

12.

Boujendar

Remacle

Hill

. Taurine supplementation to the low protein maternal diet restores a normal development of the endocrine pancreas in the offspring. Diabetologia 2000; 43 (Suppl 1): A128.

13.

Cherif

Reusens

Ahn

. Effects of taurine on the insulin secretion of rat fetal islets from dams fed a low-protein diet. J Endocrinol 1998; 159: 341–348.

14.

Xavier

Magalhães

Gontijo

. Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats. Braz J Med Biol Res 2000; 33: 347–354.

15.

Furlan

Marshall

Macedo

. Acute intracerebroventricular insulin microinjection after nitric oxide synthase inhibition of renal sodium handling in rats. Life Sci 2003; 72: 2561–2569.

16.

Boer

Morelli

Figueiredo

. Early altered renal sodium handling determined by lithium clearance in spontaneously hypertensive rats (SHR): Role of renal nerves. Life Sci 2005; 76: 1805–1815.

17.

Guadagnini

Gontijo

. Altered renal sodium handling in spontaneously hypertensive rats (SHR) after hypertonic saline intracerebroventricular injection: Role of renal nerves. Life Sci 2006; 79: 1666–1673.

18.

Herculano-Houzel

Lent

. Isotropic fractionator: A simple, rapid method for the quantification of total cell and neuron numbers in the brain. J Neurosci 2005; 25: 2518–2521.

19.

Mesquita

Gontijo

Boer

. Expression of renin-angiotensin system signaling compounds in maternal protein-restricted rats: Effect on renal sodium excretion and blood pressure. Nephrol Dial Transplant 2010; 25: 380–388.

20.

Blair

Stanley

. Intrauterine growth and spastic cerebral palsy. I. Association with birth weight for gestational age. Am J Obstet Gynecol 1990; 162: 229–237.

21.

Fanaroff

Wright

Stevenson

. Very-low-birth-weight outcomes of the National Institute of Child Health and Human Development Neonatal Research Network, May 1991 through December 1992. Am J Obstet Gynecol 1995; 173: 1423–1431.

22.

Mesquita

Gontijo

Boer

. Maternal undernutrition and the offspring kidney: From fetal to adult life. Braz J Med Biol Res 2010; 43: 1010–1018.

23.

Matos

Orozco-Solís

Lopes

Souza

. Nutrient restriction during early life reduces cell proliferation in the hippocampus at adulthood but does not impair the neuronal differentiation process of the new generated cells. Neurosci 2011; 196: 16–24.

24.

Healy

Rettig

Nguyen

. Autoradiographic evidence that angiotensin II receptors are associated with vagal afferents and efferents within the solitary-vagal area of the rat brainstem. J Hypertens 1986; 4 (Suppl 6): S462–S464.

25.

Szigethy

Barnes

Diz

. Light microscopic localization of angiotensin II binding sites in canine medulla using high resolution autoradiography. Brain Res Bull 1992; 29: 813–819.

26.

Barnes

McQueeney

Ferrario

. Receptor subtype that mediates the neuronal effects of angiotensin II in the rat dorsal medulla. Brain Res Bull 1993; 31: 195–200.

27.

Sumal

Blessing

Joh

. Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius. Brain Res 1983; 277: 31–40.

28.

Helke

. Neuroanatomical localization of substance P: Implications for central cardiovascular control. Peptides 1982; 3: 479–483.

29.

Maley

. The ultrastructural localization of enkephalin and substance P immunoreactivities in the nucleus tractus solitarii of the cat. J Comp Neurol 1985; 233: 490–496.

30.

Gatti

Shirahata

Johnson

. Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat. Brain Res 1995; 693: 133–147.

31.

Unger

. The angiotensin type 2 receptor: Variations on an enigmatic theme. J Hypertens 1990; 17 (12 Pt 2): 1775–1786.

32.

Mathai

Evered

McKinley

. Central losartan blocks natriuretic, vasopressin, and pressor responses to central hypertonic NaCl in sheep. Am J Physiol Renal Physiol 1998; 275: R548-R554.

33.

Giles

Sly

McKinley

. Neurons in the lamina terminalis which project polysynaptically to the kidney express angiotensin AT1A receptor. Brain Res 2001; 898: 9–12.

34.

Giles

Fernley

Nakamura

. Characterization of a specific antibody to the rat angiotensin II AT1 receptor. J Histochem Cytochem 1999; 47: 507–516.

35.

Zapparoli

Figueiredo

Boer

. Impaired dipsogenic and renal response to repetitive intracerebroventricular angiotensin II (AngII) injections in rats. J Renin Angiotensin Aldosterone Syst 2011; 12: 161–168.

36.

Torsoni

Carvalheira

Calegari

. Angiotensin II (AngII) induces the expression of suppressor of cytokine signaling (SOCS)-3 in rat hypothalamus — a mechanism for desensitization of AngII signaling. J Endocrinol 2004; 181: 117–128.

37.

Yamada

Akishita

Ito

. AT2 receptor and vascular smooth muscle cell differentiation in vascular development. Hypertension 1999; 33: 1414–1419.

38.

Mogi

Tsukuda

. Angiotensin II-induced neural differentiation via angiotensin II type 2 (AT2) receptor-MMS2 cascade involving interaction between AT2 receptor-interacting protein and Src homology 2 domain-containing protein-tyrosine phosphatase 1. Mol Endocrinol 2007; 21: 499–511.

39.

Höhle

Blume

Lebrun

. Angiotensin receptors in the brain. Pharmacol Toxicol 1995; 77: 306–315.

40.

Philipps

Holzman

Teng

. Tissue concentrations of free amino acids in term human placentas. Am J Obstet Gynecol 1978; 131: 881–887.

41.

Miyamoto

Balkovetz

Leibach

. Na⁺Cl^– gradient-driven, high affinity, uphill transport of taurine in human placental brush-border membrane vesicles. FEBS Lett 1988; 231: 263–267.

42.

Gaull

Sturman

Räihä

NCR

. Development of mammalian sulfur metabolism: Absence of cystathionase in human fetal tissue. Pediatr Res 1972; 6: 538–547.

43.

Wang

J-H

Redmond

Watson

. The beneﬁcial effect of taurine on the prevention of human endothelial cell death. Shock 1996; 6: 331–338.

44.

Ballard

Schaffer

. Stimulation of the Na/Ca exchanger by phenylephrine, angiotensin II and endothelin 1. J Mol Cell Cardiol 1996; 28: 11–17.

45.

Fukuta

Yoshizumi

Kitagawa

. Effect of angiotensin II on Ca efﬂux from freshly isolated adult rat cardiomyocytes: Possible involvement of Na/Ca exchanger. Biochem Pharmacol 1998; 55: 481–487.

46.

Schaffer

Lombardini

Azuma

. Interaction between the actions of taurine and angiotensin II. Amino Acids 2000; 18: 305–318.

47.

Kohashi

Okabayashi

Hama

. Decreased urinary taurine in essential hypertension. Prog Clin Biol Res 1983; 125: 73–87.

48.

Yamamoto

Akabane

Yoshimi

. Effects of taurine on stress-evoked hemodynamic and plasma catecholamine changes in spontaneously hypertensive rats. Hypertension 1985; 7: 913–922.

49.

Fujita

Sato

. Hypotensive effect of taurine. Possible involvement of the sympathetic nervous system and endogenous opiates. J Clin Invest 1988; 82: 993–997.

50.

Ideishi

Miura

Sakai

. Taurine amplifies renal kallikrein and prevents salt-induced hypertension in Dahl rats. J Hypertens 1994; 12: 653–661.

51.

Paxinos

Watson

. The Rat Brain in Stereotaxic Coordinates. 6th Edition, 2006; London, UK: Academic Press.

Impact of taurine supplementation on blood pressure in gestational protein-restricted offspring: Effect on the medial solitary tract nucleus cell numbers,angiotensin receptors,and renal sodium handling

Abstract

Objective:

Methods and results:

Conclusion:

Keywords

Introduction

Materials and methods

Animals

Blood pressure measurement

Total cell and neuron quantification of the medulla oblongata

Renal function evaluation

Western blot

Tissue processing, histology and immunohistochemical procedures

Antibodies and chemicals

Data presentation and statistical analysis

Results

Total cell and neuron quantification of the medulla oblongata

Renal function data

Western blot analysis of AT1R and AT2R expression

nTS immunohistochemical analysis of AT1R and AT2R

Discussion

Conclusion

Footnotes

Acknowledgements

Conflict of interest

Funding

References

Western blot analysis of AT1_R and AT2_R expression

nTS immunohistochemical analysis of AT₁R and AT₂R