Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero
1 Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK
Abstract
In the rat, protein restriction during pregnancy increases offspring blood pressure by 20–30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo. Hence, we hypothesized that exposure to a maternal low-protein diet increases glomerular Ang II AT1 receptor expression and decreases AT2 receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT1 and AT2 receptor expression, 125I-Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT1 receptor expression was increased (62%) and AT2 expression was decreased (35%) in LP rats. Maximum 125I-Ang II (125I-Ang II) binding (Bmax) was increased in LP rats (control n = 9, 291.6 ± 27.4 versus LP n = 7, 445.7 ± 27.4 fmol (mg glomerular protein)–1, P < 0.01), but affinity (KD) was not statistically different from controls (control 2.87 ± 0.85 versus LP 0.84 ± 0.20 pmol 125I-Ang II, P = 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT1 receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo. This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.
, 百拇医药
Introduction
A number of dietary interventions during pregnancy in the rat, including protein restriction (Langley & Jackson, 1994), iron restriction (Lewis et al. 2002) or feeding a high fat diet (Khan et al. 2003) to the dam, have been reported to result in increased offspring blood pressure. The most commonly used model is the maternal low-protein rat. A modest reduction in protein intake from the 12% protein minimum required by a pregnant rat (Clarke et al. 1977) to a 9% protein intake for as little as 1 week of pregnancy (gestation lasts 22 days in the rat) results in a 20–30 mmHg increase in offspring systolic blood pressure (Langley-Evans et al. 1996b). This increase in blood pressure lasts for at least 21 weeks (Langley & Jackson, 1994), and probably for the whole life of the rat.
, 百拇医药
A number of mechanisms have been proposed to account for the in utero programming effect of protein restriction. These include increased placental transfer of maternal glucocorticoids (Langley-Evans, 1997), increased sensitivity of the hypothalamo–pituitary–adrenal axis (Langley-Evans et al. 1996a) and, more recently, inhibition of nephrogenesis through suppression of the renin–angiotensin system (Woods et al. 2001). The latter hypothesis suggests that an intrauterine insult reduces renal renin–angiotensin system activity in the neonate, inhibiting nephrogenesis. This reduction in nephron number leads to a fall in total glomerular filtration rate (GFR), but an increase in single nephron GFR, which eventually leads to renal disease and hypertension (Woods et al. 2001).
, 百拇医药
However, there is also evidence which suggests that the renin–angiotensin system is upregulated in the young low-protein (LP) rat. Administration of the converting enzyme inhibitor captopril (Sherman & Langley-Evans, 1998) or the AT1 receptor antagonist losartan (Sherman & Langley-Evans, 2000) from 2 to 4 weeks of age lowered long-term blood pressure in the LP rat. Similar treatment with the Ca2+ channel antagonist nifedipine was ineffective (Sherman & Langley-Evans, 2000), suggesting that the hypertension was mediated by the renin–angiotensin system. We have reported that 4-week-old LP rats responded to a nonpressor dose of angiotensin II (Ang II) with a far greater reduction in GFR than control rats (Sahajpal & Ashton, 2003). Western blot analysis revealed increased AT1 receptor expression in the kidneys of LP rats (Sahajpal & Ashton, 2003). Together, these observations suggest that at 4 weeks of age, glomerular AT1 receptor expression is increased in LP rats. This could lead to a reduction in GFR and a subsequent increase in blood pressure.
, http://www.100md.com
The rat expresses two AT1 receptor subtypes, AT1A and AT1B, both of which are found in afferent arterioles, mesangial cells and some efferent arterioles (Ardaillou et al. 1999; Helou et al. 2003), although the former tend to be more abundant (Ruan et al. 1997). Renal AT1 receptor expression is not limited to the glomerulus; AT1 receptors are also found in the proximal tubule, thick ascending limb, distal tubule and collecting duct, as well as the afferent and efferent arterioles, interlobar and arcuate arteries, and the descending vasa recta (Miyata et al. 1999). Hence, despite the evidence from our in vivo study which suggests that the increased AT1 expression is located in the glomerulus, we could not be certain as we used whole kidney homogenates for our Western blot analyses (Sahajpal & Ashton, 2003).
, 百拇医药
The kidney also expresses the AT2 receptor. Its primary role appears to be in the control of organogenesis, and it is widely expressed within the fetal kidney (Shanmugam et al. 1995). However, it has also been observed at lower levels in the adult kidney (Ruan et al. 1997), and has been localized to the proximal tubule, collecting duct, arcuate arteries and afferent arterioles (Miyata et al. 1999). It appears to counterbalance the vasoconstrictor actions of Ang II mediated via the AT1 receptor by inducing nitric oxide production (Siragy & Carey, 1997). Hence, a reduction in AT2 expression or an imbalance in the expression of AT1 and AT2 receptors could alter renal haemodynamics in the LP rat.
, http://www.100md.com
We hypothesized that an increase in the expression of glomerular AT1 receptors and/or a reduction in AT2 receptors could account for the enhanced renal haemodynamic sensitivity of LP rats to Ang II in our in vivo study (Sahajpal & Ashton, 2003). Accordingly, the aim of this study was to determine cortical AT1 and AT2 receptor expression, and specific Ang II binding to isolated glomeruli, from LP and control rats. We also measured renal renin activity, tissue Ang II and plasma aldosterone concentrations to determine whether altered AT1 and AT2 receptor expression was associated with suppressed renin–angiotensin system activity.
, 百拇医药
Methods
All experiments described herein using animals were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, and they received local ethical approval.
Animals
Forty-two female Wistar rats (Harlan UK Limited) were used to generate the experimental animals. The dams were paired with a male in individual breeding cages, and held in a room at 22–24°C with a 12 h light:12 h dark cycle. As soon as mating was confirmed by the presence of a plug, the female's diet was switched from standard chow (Rat & Mouse Standard Diet, Bantin & Kingman Ltd, Hull, North Humberside, UK) to one which contained either 9% protein (LP) or 18% protein (control) (Table 1), as previously described (Sahajpal & Ashton, 2003). On the day of birth, litters were culled to eight pups, and left undisturbed thereafter. The dam's diet reverted to standard chow, and the offspring were subsequently weaned onto the same diet. Male offspring were studied at the age of 4 weeks.
, http://www.100md.com
Renal AT1 and AT2 receptor expression
Kidneys were harvested from 4-week-old rats, which had been killed by dislocation of the neck (control n = 5 from five litters, LP n = 5 from five litters), for Western blot analysis of AT1 and AT2 receptors. Each kidney was bisected longitudinally, and the medulla removed to leave only the cortex. Membranes were isolated by homogenization and centrifugation, as previously described (Sahajpal & Ashton, 2003). The protein concentration of the resuspended membrane pellet was determined using the Bradford method (Bio-Rad Assay Reagent, Bio-Rad Laboratories, Hercules, CA, USA). Tissue homogenate (50 μg) was then denatured at 80°C for 5 min, and fractionated using an 8% SDS-polyacrylamide gel. Blots were incubated with primary antibody (1:300, affinity-purified rabbit polyclonal AT1 (N-10) or goat polyclonal AT2 (C-18), Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), horseradish-peroxidase-conjugated anti-rabbit secondary antibody (1:5000), and developed with the ECL plus detection kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), as previously described (Sahajpal & Ashton, 2003). Immunoreactivity was quantified by densitometry, and normalized to the control group bands. Within each gel, the mean density of the control group bands was taken to represent 100%; individual densitometry readings for both control and LP bands were then expressed as a percentage of this value, allowing the calculation of standard errors.
, 百拇医药
Glomerular ligand binding assay
Ang II binding was determined in glomeruli isolated from a separate group of 4-week-old animals killed by dislocation of the neck, as previously described (Messenger et al. 1988). Excised kidneys were placed in ice-cold PBS (phosphate-buffered saline, pH 7.4), bisected longitudinally and the medulla removed. The outer cortex was cut into 1 mm cubes, minced and aspirated through a 100 μl pipette tip. The resultant homogenate was washed with 12 ml of ice-cold PBS through graded nylon mesh filters (Cell Micro Sieve, Bio-Design, Inc., New York, USA) of 200 μm and then 100 μm pore size. After centrifugation at 120 g for 5 min at 4°C, the pellet was resuspended in 1 ml of ice-cold PBS, and passed through a 70 μm pore size nylon mesh filter with a further 4 ml of buffer. Glomeruli were recovered at the top of the last sieve by rinsing with PBS. The suspension was centrifuged again at 120 g for 5 min at 4°C, resuspended in PBS, and stored at –80°C. The final suspension was >80% pure and contained isolated, intact glomeruli.
, http://www.100md.com
Saturable binding was assessed using 125I-labelled Ang II (Amersham Pharmacia Biotech) at concentrations from 10–10 to 10–7 M (control n = 9 from six litters, LP n = 7 from six litters). Selective displacement of 125I-Ang II from AT1 and AT2 receptors was demonstrated in a separate series of binding assays by coincubation with losartan (10–4 M; Merck Sharp & Dohme Ltd, Hoddesdon, Herts, UK; control n = 7 from five litters, LP n = 7 from five litters), PD123319 (10–4 M; Sigma, Poole, Dorset, UK; control n = 7 from five litters, LP n = 7 from five litters), or a combination of losartan (10–4 M) and PD123319 (10–4 M) (control n = 4 from four litters, LP n = 4 from four litters). Incubations were carried out in duplicate at room temperature in bovine-serum-albumin-coated tubes. The incubation medium was made up to a total volume of 200 μl consisting of PBS (pH 7.4), the glomerular suspension (equivalent to 20 μg protein) and 125I-labelled Ang II. Non-specific binding was determined by the amount of 125I-labelled Ang II bound in the presence of excess unlabelled Ang II (5 x 10–5 M). Total activity was also determined for each 125I-labelled Ang II concentration. After incubation for 45 min at room temperature, samples were centrifuged at 10 000 g for 10 min at room temperature, resuspended in 1 ml PBS and centrifuged again. Bound activity was determined using a Cobra II gamma counter (Packard, Pangbourne, Berkshire, UK). Specific binding is expressed as femtomoles of bound Ang II per milligram of glomerular protein. The binding affinity constant (KD) and binding capacity (Bmax) were derived by Scatchard transformation of binding data (GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, CA, USA).
, 百拇医药
Renal renin radioimmunoassay
Kidneys were collected from control (n = 9 from five litters) and LP rats (n = 7 from five litters), which had been killed by dislocation of the neck. Tissue was homogenized on ice in Tris/HCl buffer (pH 7.4, 1 ml buffer per 0.1 g tissue) containing enzymatic inhibitors (1% Triton X-100, 2 mM EDTA, 1 μg ml–1 leupeptin, 1 μg ml–1 aprotinin, 0.1 μg ml–1 phenylmethylsulphonyl fluoride and 0.1 μg ml–1 bacitracin), and centrifuged at 1000 g for 20 min at 4°C. Samples were diluted in 0.5 M phosphate buffer (pH 6.5) containing 8.8 mM EDTA, 3 mM 8-hydroxyquinoline sulphate and 5 mM 2,3-dimercaptopropanol to a final concentration of 1:4000 using a two-step dilution; in the second step 100 μl of renin-free plasma was added. Renal renin activity was measured using a REN-CT2 kit (CIS (UK) Limited, High Wycombe, Buckinghamshire, UK), as previously described (Gouldsborough et al. 2003). The kit includes an angiotensin converting enzyme inhibitor solution (specific details of its formulation are not provided by the manufacturer) to prevent degradation of angiotensin I (Ang I) during incubation of samples. Renin activity is expressed in micrograms of Ang I per gram kidney weight per hour.
, 百拇医药
Renal Ang II radioimmunoassay
Kidneys were collected from control (n = 5 from five litters) and LP rats (n = 5 from five litters), which had been killed by dislocation of the neck. A 75 μl aliquot of homogenized kidney, prepared as above, was added to 25 μl assay buffer containing captopril (5 ng mg–1 of tissue) to inhibit Ang II generation in vitro. Ang II was measured by radioimmunoassay, as previously described (Ashton & Balment, 1991), using 3-[125I]iodotyrosyl4 Ang II (5-isoleucine) (2000 Ci mmol–1) and rabbit Ang II antibody (Amersham Pharmacia Biotech). Tissue Ang II concentration is expressed in picomoles per gram kidney weight.
, 百拇医药
Plasma aldosterone radioimmunoassay
Blood from control (n = 5 from five litters) and LP rats (n = 5 from 5 litters), which had been killed by stunning followed by decapitation, was collected into ice-cold heparinized tubes. The plasma was separated by centrifugation, and assayed for aldosterone using a commercial radioimmunoassay kit (Coat-a-Count, Diagnostic Products Corporation, Caernarfon, Gwynedd, UK). The intra-assay coefficient of variation was 3.2%.
, 百拇医药
Statistical analysis
All data are presented as means ± S.E.M. Statistical analysis was by Student's unpaired t test or one-way ANOVA and Duncan's test, as appropriate, with significance ascribed at the 5% level (SPSS for Windows, version 11.5.0, SPSS UK Ltd, Surrey, UK). Saturation curves, binding constants (Bmax) and binding affinity constants (KD) were calculated using GraphPad Prism (version 3.02 for Windows, GraphPad Software). Best fit for one- versus two-site binding equations was compared by F test. The simpler equation was accepted when P > 0.05.
, 百拇医药
Results
Western blot analysis of the kidney cortex from control and LP rats yielded a single band at the expected weight of 45 kDa, corresponding to the Ang II AT1 receptor (Fig. 1). Incubation in the presence of excess immunizing peptide completely blocked antibody binding. AT1 expression in the cortex of 4-week-old LP rats was 62% greater (P < 0.05) than that in control rats. Western blot analysis of the AT2 receptor yielded a band at the expected weight of 45 kDa (Fig. 2). In contrast to the level of AT1 expression, cortical Ang II AT2 receptor expression was significantly lower (P < 0.05) by 35% in LP rats.
, 百拇医药
A, typical immunoblot for the AT1 receptor in the renal cortex of 4-week-old control (C) and low-protein (LP) rats. B, renal cortical AT1 receptor expression in 4-week-old control (open bars, n = 5 from 5 litters) and LP rats (filled bars, n = 5 from 5 litters). Data normalized to the control bands and presented as means ± S.E.M. Statistical comparisons were by Student's unpaired t test. *P < 0.05 control versus LP.
A, typical immunoblot for the AT2 receptor in the renal cortex of 4-week-old control and LP rats. B, renal cortical AT2 receptor expression in 4-week-old control (open bars, n = 5 from 5 litters) and LP rats (filled bars, n = 5 from 5 litters). Data normalized to the control bands and presented as means ± S.E.M. Statistical comparisons were by Student's unpaired t test. *P < 0.05 control versus LP.
, 百拇医药
Figure 3 shows saturation curves of 125I-Ang II binding to glomeruli from LP and control rats in the presence of either the AT1 receptor antagonist losartan or the AT2 receptor antagonist PD123319. One- and two-binding site equations were applied to the saturation curves for 125I-Ang II binding alone, and the goodness of fit compared. For both the control and LP rat binding curves, the one-site model provided the best fit (one versus two-site: control F2,3 = 3.734 P = 0.153; LP F2,3 = 0.976 P = 0.471), therefore the Bmax and KD data have been calculated on this basis. The increase in LP rat AT1 receptor expression detected by Western blotting was reflected by a significant increase in 125I-Ang II binding by the isolated glomeruli. Bmax for LP rats was significantly higher (P < 0.01) than that for control rats (Table 2). KD tended to be lower in LP rats, but this did not reach statistical significance (P = 0.059).
, 百拇医药
A, saturation curves of 125I-Ang II binding to glomeruli isolated from 4-week-old control and LP rats. Binding curves are shown for 125I-Ang II alone (, control n = 9 from 6 litters, , LP n = 7 from 6 litters), 125I-Ang II and losartan (, control n = 7 from 5 litters, , LP n = 7 from 5 litters) and 125I-Ang II and PD123319 ( , control n = 7 from 5 litters, , LP n = 7 from 5 litters). B, Scatchard plots for 125I-Ang II binding alone. Correlation coefficients for the Scatchard plots were significant for both control (r = –0.771, P < 0.05) and LP rats (r = –0.916, P < 0.01).
, 百拇医药
Co-incubating glomeruli with 125I-Ang II and losartan significantly reduced (P < 0.001), but did not completely abolish, 125I-Ang II binding (Fig. 3). Losartan reduced the Bmax of LP rats by 94%, compared with an 85% reduction in control rats (Table 2). Net losartan-sensitive binding in LP rats amounted to 419.4 ± 15.6 compared with 247.2 ± 16.6 fmol (mg glomerular protein)–1 in control animals (P < 0.05). KD values remained similar to those observed for 125I-Ang II alone, and did not differ between LP and control rats. Co-incubation of glomeruli with 125I-Ang II and PD123319 also significantly reduced (P < 0.05) Bmax in both control and LP rats, but to a lesser extent than that seen with losartan. PD12319-sensitive binding was equivalent to 74.2 ± 21.9 and 75.2 ± 23.6 fmol (mg glomerular protein)–1 in control and LP rats, respectively. KD values remained similar to those observed for 125I-Ang II alone; the KD of control rats was significantly higher than that of LP rats (P < 0.05, Table 2). 125I-Ang II binding was only completely abolished by coincubation with losartan and PD123319 (counts not significantly different from background at 10–10–10–7 M Ang II).
, http://www.100md.com
Renal renin activity (Fig. 4A, P = 0.051), renal tissue Ang II (Fig. 4B, P = 0.97) and plasma aldosterone concentrations (Fig. 4C, P = 0.18) did not differ between control and LP rats.
A, renal renin activity (micrograms of angiotensin I per gram kidney weight per hour) in kidneys from 4-week-old control (open bars, n = 9 from 5 litters) and LP (filled bars, n = 7 from 5 litters) rats. B, renal Ang II concentrations (picomoles per gram kidney weight) in kidneys from 4-week-old control (open bars, n = 5 from 5 litters) and LP (filled bars, n = 5 from 5 litters) rats. C, plasma aldosterone concentrations (picomoles per litre) in 4-week-old control (open bars, n = 5 from 5 litters) and LP (filled bars, n = 5 from 5 litters) rats. Statistical comparisons were by Student's unpaired t test.
, 百拇医药
Discussion
This study set out to test the hypothesis that exposure to a maternal LP diet in utero increases expression of glomerular AT1 receptors, and reduces AT2 receptor expression in the young rat. This would account for our previous observations of enhanced glomerular sensitivity to Ang II in LP rats in vivo (Sahajpal & Ashton, 2003), which may contribute to the increase in blood pressure seen in these rats. It provides evidence of an increase in renal AT1 receptor expression and, for the first time, a specific increase in losartan-sensitive Ang II binding capacity in the glomeruli of these rats. These observations are consistent with a recent report (Vehaskari et al. 2004) showing increased AT1 protein and AT1A mRNA expression at 4 weeks of age in a similar, but not identical, model of maternal protein restriction. Furthermore, the increase in AT1 receptor expression observed herein was coupled with a reduction in AT2 receptor expression, which is consistent with a recent report of reduced renal AT2 mRNA in LP rats of a similar age (McMullen et al. 2004). In contrast to earlier observations in neonatal LP rats (Woods et al. 2001), renal renin activity and tissue Ang II concentrations were not lower in LP rats at 4 weeks of age. These data provide evidence that the renin–angiotensin system is not suppressed in the LP rat at this stage of development.
, 百拇医药
There is some published information on the effect of a maternal LP diet on offspring renal renin–angiotensin system activity in the rat, but this is confused by the use of different dietary protein restriction protocols. Renal tissue renin, renin mRNA and Ang II concentrations have been reported to be reduced in LP rats at postnatal days 1 and 5 (Woods et al. 2001). Plasma renin activity has been shown to be reduced in LP rats at 4 weeks of age (Vehaskari et al. 2001), but return to normal at 13 weeks (Langley-Evans & Jackson, 1995). Plasma angiotensin converting enzyme (ACE) activity is increased at 4 weeks, but lung and renal ACE activity are not different (Langley-Evans & Jackson, 1995; Nwagwu et al. 2000). However, apart from our earlier study (Sahajpal & Ashton, 2003) and the recent report by Vehaskari et al. (2004), renal AT1 receptor expression has not been described in LP rats.
, 百拇医药
The renin–angiotensin system plays a critical role in renal development (Guron & Friberg, 2000). This is reflected by the widespread distribution of renin and both AT1 and AT2 receptors within the fetal and neonatal kidney (Gomez et al. 1993). Immunohistochemical analysis has shown that renin immunoreactivity is widespread in the neonatal kidney, being located along the entire length of the afferent arteriole and interlobar artery (Gomez et al. 1989). This contrasts with the adult pattern of distribution in which renin-secreting cells are restricted to the juxtaglomerular position (Gomez et al. 1989). Consistent with its wider distribution in the neonatal kidney, renin mRNA and renal tissue renin activity are also greater in the neonatal kidney by comparison with adult rats (Gomez et al. 1989). In LP rats, renal renin activity is very low in the newborn, relative to controls (Woods et al. 2001), but by 4 weeks of age renin activity and renal Ang II concentrations are comparable with control animals. Similar observations have been reported in another model of maternal protein restriction. Plasma renin activity was low at birth, but gradually increased to exceed that of controls in adulthood (Manning & Vehaskari, 2001). Plasma and renal Ang I and Ang II concentrations were similar (Vehaskari et al. 2004) whereas plasma aldosterone concentrations were elevated (Vehaskari et al. 2001) in LP rats compared with controls at 4 weeks of age. These observations are in broad agreement with our own: renal renin activity, renal Ang II and plasma aldosterone concentrations were all similar in 4-week-old LP and control rats. Taken together, these data suggest that the renin–angiotensin system is no longer suppressed in the LP rat at this stage of development.
, 百拇医药
Renal AT1 receptors first appear in mesenchymal cells and differentiating glomeruli at embryonic day E15 in the rat (Shanmugam et al. 1994). AT1 mRNA increases fivefold from E17 to postnatal day 5 (Tufro-McReddie et al. 1993b), reaching a plateau at around postnatal day 10–20 (Aguilera et al. 1994). AT1 receptors are located throughout the nephrogenic cortex of newborn kidneys, as well as in glomeruli, tubules and vessels (Tufro-McReddie et al. 1993b), the AT1A subtype being the predominant form (Shanmugam et al. 1994). As the kidney matures, the adult pattern of receptor distribution appears, which comprises the glomeruli, proximal tubule, thick ascending limb, distal tubule and collecting duct, as well as the afferent and efferent arterioles, interlobar and arcuate arteries and the descending vasa recta (Miyata et al. 1999). Hence renal AT1 receptor expression in the adult kidney is located primarily in the cortex. The ligand-binding data described herein demonstrate for the first time that 125I-Ang II binding is enhanced in glomeruli from LP rats. Losartan-sensitive binding was 70% higher in LP rat glomeruli, which is consistent with the 62% increase in AT1 receptor expression detected by Western blotting of the renal cortex. These data are also in agreement with previously reported increases in both AT1 receptor protein and mRNA in whole kidney homogenates (Sahajpal & Ashton, 2003; Vehaskari et al. 2004). Therefore, the data presented in the current study are consistent with an increase in AT1 receptor expression in the renal cortex of LP rats, with a specific increase in glomerular receptors. An increase in proximal tubular AT1 receptor expression is unlikely, as Ang-II-mediated Na+ reabsorption did not differ in vivo between LP and control rats (Sahajpal & Ashton, 2003), but we cannot rule out the possibility that AT1 receptors are upregulated in other parts of the nephron or the renal vasculature.
, 百拇医药
In contrast to the AT1 receptor, renal AT2 receptor expression is maximal during nephrogenesis and declines once the kidney matures (Miyata et al. 1999). In the immature kidney, AT2 receptors are located in the nephrogenic zone of the metanephric cortex and ureteric bud (Aguilera et al. 1994). In adult rats, AT2 receptors have been localized to the proximal tubule, collecting duct, arcuate arteries and afferent arterioles (Miyata et al. 1999). There are conflicting reports over AT2 receptor expression in glomeruli (Ozono et al. 1997; Miyata et al. 1999), but functional studies suggest that AT2 receptors can influence the glomerular filtration rate through a vasodilator action (Siragy & Carey, 1997). In the current study, coincubation of glomeruli with 125I-Ang II and PD123319 (AT2 antagonist) significantly reduced the Bmax of both control and LP rats by 17 and 25%, respectively. Furthermore, Ang II binding could not be totally blocked by losartan (AT1 antagonist); a combination of losartan and PD123319 was required to completely block Ang II binding. This suggests that there are low levels of AT2 expression in the glomeruli of both groups. Taking the ligand binding data together with the reduction in AT2 receptor expression observed herein by Western blotting and the reported reduction in AT2 mRNA (McMullen et al. 2004), these observations suggest that the balance between vasoconstriction and vasodilatation is tipped towards constriction in the young LP rat. This is consistent with our earlier observation of a greater reduction in GFR in response to a nonpressor dose of Ang II in the LP rat (Sahajpal & Ashton, 2003).
, 百拇医药
The underlying mechanisms responsible for the increase in AT1 and a decrease in AT2 expression in 4-week-old LP rats are not clear. Blood pressure is already elevated at this age (Sahajpal & Ashton, 2003), which should suppress AT1 expression and renin activity (Tufro-McReddie et al. 1993a). Newborn LP rats have low renal renin activity (Woods et al. 2001), which might be expected to increase glomerular AT1 receptor expression (Wilkes et al. 1988). However, by 4 weeks of age, renal renin activity was comparable between control and LP rats, suggesting that regulation of the renin–angiotensin system changes as the LP rat matures. Increased exposure to maternal glucocorticoids, as a result of placental 11hydroxysteroid dehydrogenase type 2 suppression, has been linked to programming of the renin–angiotensin system in the LP rat (Langley-Evans et al. 1999; Woods, 2000). Clearly, further study is required before the mechanisms involved are fully understood.
, 百拇医药
In summary, this study has confirmed the hypothesis that maternal protein restriction in the rat results in an increase in AT1 receptor expression, coupled with a reduction in AT2 receptors, in male offspring at 4 weeks of age. This was associated with renal tissue renin activity, tissue Ang II and plasma aldosterone concentrations comparable with control animals. The mechanisms responsible for this programming action on the renal renin–angiotensin system are not yet clear, but may be linked to exposure of the fetus to maternal glucocorticoids. The consequence of increased AT1 receptor expression in the glomeruli of LP rats is an increased sensitivity to Ang II, resulting in an inappropriate reduction in GFR. This may be exacerbated by the reduction in nephron number reported in these animals (Sahajpal & Ashton, 2003), leading to a further reduction in the overall filtration capacity of the LP rat kidney, retention of salt and water and eventually an increase in blood pressure.
, 百拇医药
References
Aguilera G, Kapur S, Feuillan P, Sunar-Akbasak B & Bathia AJ (1994). Developmental changes in angiotensin II receptor subtypes and AT1 receptor mRNA in rat kidney. Kidney Int 46, 973–979.
Ardaillou R, Chansel D, Chatziantoniou C & Dussaule JC (1999). Mesangial AT1 receptors: expression, signaling, and regulation. J Am Soc Nephrol 10 (Suppl. 11), S40–S46.
Ashton N & Balment RJ (1991). Sexual dimorphism in renal function and hormonal status of New Zealand genetically hypertensive rats. Acta Endocrinol 124, 91–97.
, http://www.100md.com
Clarke HE, Coates ME, Eva JK, Ford DJ, Milner CK, O'Donoghue PN, Scott PP & Ward RJ (1977). Dietary standards for laboratory animals: report of the Laboratory Animals Centre Diets Advisory Committee. Lab Anim 11, 1–28.
Gomez RA, Lynch KR, Sturgill BC, Elwood JP, Chevalier RL, Carey RM & Peach MJ (1989). Distribution of renin mRNA and its protein in the developing kidney. Am J Physiol Renal Physiol 257, F850–858.
Gomez RA, Tufro-McReddie A, Everett AD & Pentz ES (1993). Ontogeny of renin and AT1 receptor in the rat. Pediatr Nephrol 7, 635–638.
, 百拇医药
Gouldsborough I, Lindop GB & Ashton N (2003). Renal renin–angiotensin system activity in naturally reared and cross-fostered spontaneously hypertensive rats. Am J Hypertens 16, 864–869.
Guron G & Friberg P (2000). An intact renin–angiotensin system is a prerequisite for normal renal development. J Hypertens 18, 123–137.
Helou CM, Imbert-Teboul M, Doucet A, Rajerison R, Chollet C, Alhenc-Gelas F & Marchetti J (2003). Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney. Am J Physiol Renal Physiol 285, F507–514.
, 百拇医药
Khan IY, Taylor PD, Dekou V, Seed PT, Lakasing L, Graham D, Dominiczak AF, Hanson MA & Poston L (2003). Gender-linked hypertension in offspring of lard-fed pregnant rats. Hypertension 41, 168–175.
Langley SC & Jackson AA (1994). Increased systolic pressure in adult rats induced by fetal exposure to maternal low protein diet. Clin Sci 86, 217–222.
Langley-Evans SC (1997). Intrauterine programming of hypertension by glucocorticoids. Life Sci 60, 1213–1221.
, 百拇医药
Langley-Evans SC, Gardner DS & Jackson AA (1996a). Maternal protein restriction influences the programming of the rat hypothalamic–pituitary–adrenal axis. J Nutr 126, 1578–1585.
Langley-Evans SC & Jackson AA (1995). Captopril normalises systolic blood pressure in rats with hypertension induced by fetal exposure to maternal low protein diets. Comp Biochem Physiol A Physiol 110, 223–228.
Langley-Evans SC, Sherman RC, Welham SJ, Nwagwu MO, Gardner DS & Jackson AA (1999). Intrauterine programming of hypertension: the role of the renin–angiotensin system. Biochem Soc Trans 27, 88–93.
, 百拇医药
Langley-Evans SC, Welham SJ, Sherman RC & Jackson AA (1996b). Weanling rats exposed to maternal low-protein diets during discrete periods of gestation exhibit differing severity of hypertension. Clin Sci 91, 607–615.
Lewis RM, Forhead AJ, Petry CJ, Ozanne SE & Hales CN (2002). Long-term programming of blood pressure by maternal dietary iron restriction in the rat. Br J Nutr 88, 283–290.
Manning J & Vehaskari VM (2001). Low birth weight-associated adult hypertension in the rat. Pediatr Nephrol 16, 417–422.
, 百拇医药
McMullen S, Gardner DS & Langley-Evans SC (2004). Prenatal programming of angiotensin II type 2 receptor expression in the rat. Br J Nutr 91, 133–140.
Messenger EA, Stonier C & Aber GM (1988). Differences in glomerular binding and response to angiotensin II between normotensive and spontaneously hypertensive rats. Clin Sci 75, 191–196.
Miyata N, Park F, Li XF & Cowley AW Jr (1999). Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney. Am J Physiol Renal Physiol 277, F437–446.
, 百拇医药
Nwagwu MO, Cook A & Langley-Evans SC (2000). Evidence of progressive deterioration of renal function in rats exposed to a maternal low-protein diet in utero. Br J Nutr 83, 79–85.
Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM & Carey RM (1997). Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension 30, 1238–1246.
Ruan X, Wagner C, Chatziantoniou C, Kurtz A & Arendshorst WJ (1997). Regulation of angiotensin II receptor AT1 subtypes in renal afferent arterioles during chronic changes in sodium diet. J Clin Invest 99, 1072–1081.
, 百拇医药
Sahajpal V & Ashton N (2003). Renal function and angiotensin AT1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci 104, 607–614.
Shanmugam S, Corvol P & Gasc JM (1994). Ontogeny of the two angiotensin II type 1 receptor subtypes in rats. Am J Physiol Endocrinol Metab 267, E828–836.
Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P & Gasc JM (1995). Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland. Am J Physiol Renal Physiol 268, F922–930.
, http://www.100md.com
Sherman RC & Langley-Evans SC (1998). Early administration of angiotensin-converting enzyme inhibitor captopril, prevents the development of hypertension programmed by intrauterine exposure to a maternal low-protein diet in the rat. Clin Sci 94, 373–381.
Sherman RC & Langley-Evans SC (2000). Antihypertensive treatment in early postnatal life modulates prenatal dietary influences upon blood pressure in the rat. Clin Sci 98, 269–275.
, 百拇医药
Siragy HM & Carey RM (1997). The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest 100, 264–269.
Tufro-McReddie A, Chevalier RL, Everett AD & Gomez RA (1993a). Decreased perfusion pressure modulates renin and ANG II type 1 receptor gene expression in the rat kidney. Am J Physiol Regul Integr Comp Physiol 264, R696–702.
Tufro-McReddie A, Harrison JK, Everett AD & Gomez RA (1993b). Ontogeny of type 1 angiotensin II receptor gene expression in the rat. J Clin Invest 91, 530–537.
, 百拇医药
Vehaskari VM, Aviles DH & Manning J (2001). Prenatal programming of adult hypertension in the rat. Kidney Int 59, 238–245.
Vehaskari VM, Stewart T, Lafont D, Soyez C, Seth D & Manning J (2004). Kidney angiotensin and angiotensin receptor expression in prenatally programmed hypertension. Am J Physiol Renal Physiol 287, F262–267.
Wilkes BM, Pion I, Sollott S, Michaels S & Kiesel G (1988). Intrarenal renin–angiotensin system modulates glomerular angiotensin receptors in the rat. Am J Physiol Renal Physiol 254, F345–350.
Woods LL (2000). Fetal origins of adult hypertension: a renal mechanism Curr Opin Nephrol Hypertens 9, 419–425.
Woods LL, Ingelfinger JR, Nyengaard JR & Rasch R (2001). Maternal protein restriction suppresses the newborn renin–angiotensin system and programs adult hypertension in rats. Pediatr Res 49, 460–467., 百拇医药(Vandana Sahajpal and Nick)
Abstract
In the rat, protein restriction during pregnancy increases offspring blood pressure by 20–30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo. Hence, we hypothesized that exposure to a maternal low-protein diet increases glomerular Ang II AT1 receptor expression and decreases AT2 receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT1 and AT2 receptor expression, 125I-Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT1 receptor expression was increased (62%) and AT2 expression was decreased (35%) in LP rats. Maximum 125I-Ang II (125I-Ang II) binding (Bmax) was increased in LP rats (control n = 9, 291.6 ± 27.4 versus LP n = 7, 445.7 ± 27.4 fmol (mg glomerular protein)–1, P < 0.01), but affinity (KD) was not statistically different from controls (control 2.87 ± 0.85 versus LP 0.84 ± 0.20 pmol 125I-Ang II, P = 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT1 receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo. This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.
, 百拇医药
Introduction
A number of dietary interventions during pregnancy in the rat, including protein restriction (Langley & Jackson, 1994), iron restriction (Lewis et al. 2002) or feeding a high fat diet (Khan et al. 2003) to the dam, have been reported to result in increased offspring blood pressure. The most commonly used model is the maternal low-protein rat. A modest reduction in protein intake from the 12% protein minimum required by a pregnant rat (Clarke et al. 1977) to a 9% protein intake for as little as 1 week of pregnancy (gestation lasts 22 days in the rat) results in a 20–30 mmHg increase in offspring systolic blood pressure (Langley-Evans et al. 1996b). This increase in blood pressure lasts for at least 21 weeks (Langley & Jackson, 1994), and probably for the whole life of the rat.
, 百拇医药
A number of mechanisms have been proposed to account for the in utero programming effect of protein restriction. These include increased placental transfer of maternal glucocorticoids (Langley-Evans, 1997), increased sensitivity of the hypothalamo–pituitary–adrenal axis (Langley-Evans et al. 1996a) and, more recently, inhibition of nephrogenesis through suppression of the renin–angiotensin system (Woods et al. 2001). The latter hypothesis suggests that an intrauterine insult reduces renal renin–angiotensin system activity in the neonate, inhibiting nephrogenesis. This reduction in nephron number leads to a fall in total glomerular filtration rate (GFR), but an increase in single nephron GFR, which eventually leads to renal disease and hypertension (Woods et al. 2001).
, 百拇医药
However, there is also evidence which suggests that the renin–angiotensin system is upregulated in the young low-protein (LP) rat. Administration of the converting enzyme inhibitor captopril (Sherman & Langley-Evans, 1998) or the AT1 receptor antagonist losartan (Sherman & Langley-Evans, 2000) from 2 to 4 weeks of age lowered long-term blood pressure in the LP rat. Similar treatment with the Ca2+ channel antagonist nifedipine was ineffective (Sherman & Langley-Evans, 2000), suggesting that the hypertension was mediated by the renin–angiotensin system. We have reported that 4-week-old LP rats responded to a nonpressor dose of angiotensin II (Ang II) with a far greater reduction in GFR than control rats (Sahajpal & Ashton, 2003). Western blot analysis revealed increased AT1 receptor expression in the kidneys of LP rats (Sahajpal & Ashton, 2003). Together, these observations suggest that at 4 weeks of age, glomerular AT1 receptor expression is increased in LP rats. This could lead to a reduction in GFR and a subsequent increase in blood pressure.
, http://www.100md.com
The rat expresses two AT1 receptor subtypes, AT1A and AT1B, both of which are found in afferent arterioles, mesangial cells and some efferent arterioles (Ardaillou et al. 1999; Helou et al. 2003), although the former tend to be more abundant (Ruan et al. 1997). Renal AT1 receptor expression is not limited to the glomerulus; AT1 receptors are also found in the proximal tubule, thick ascending limb, distal tubule and collecting duct, as well as the afferent and efferent arterioles, interlobar and arcuate arteries, and the descending vasa recta (Miyata et al. 1999). Hence, despite the evidence from our in vivo study which suggests that the increased AT1 expression is located in the glomerulus, we could not be certain as we used whole kidney homogenates for our Western blot analyses (Sahajpal & Ashton, 2003).
, 百拇医药
The kidney also expresses the AT2 receptor. Its primary role appears to be in the control of organogenesis, and it is widely expressed within the fetal kidney (Shanmugam et al. 1995). However, it has also been observed at lower levels in the adult kidney (Ruan et al. 1997), and has been localized to the proximal tubule, collecting duct, arcuate arteries and afferent arterioles (Miyata et al. 1999). It appears to counterbalance the vasoconstrictor actions of Ang II mediated via the AT1 receptor by inducing nitric oxide production (Siragy & Carey, 1997). Hence, a reduction in AT2 expression or an imbalance in the expression of AT1 and AT2 receptors could alter renal haemodynamics in the LP rat.
, http://www.100md.com
We hypothesized that an increase in the expression of glomerular AT1 receptors and/or a reduction in AT2 receptors could account for the enhanced renal haemodynamic sensitivity of LP rats to Ang II in our in vivo study (Sahajpal & Ashton, 2003). Accordingly, the aim of this study was to determine cortical AT1 and AT2 receptor expression, and specific Ang II binding to isolated glomeruli, from LP and control rats. We also measured renal renin activity, tissue Ang II and plasma aldosterone concentrations to determine whether altered AT1 and AT2 receptor expression was associated with suppressed renin–angiotensin system activity.
, 百拇医药
Methods
All experiments described herein using animals were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, and they received local ethical approval.
Animals
Forty-two female Wistar rats (Harlan UK Limited) were used to generate the experimental animals. The dams were paired with a male in individual breeding cages, and held in a room at 22–24°C with a 12 h light:12 h dark cycle. As soon as mating was confirmed by the presence of a plug, the female's diet was switched from standard chow (Rat & Mouse Standard Diet, Bantin & Kingman Ltd, Hull, North Humberside, UK) to one which contained either 9% protein (LP) or 18% protein (control) (Table 1), as previously described (Sahajpal & Ashton, 2003). On the day of birth, litters were culled to eight pups, and left undisturbed thereafter. The dam's diet reverted to standard chow, and the offspring were subsequently weaned onto the same diet. Male offspring were studied at the age of 4 weeks.
, http://www.100md.com
Renal AT1 and AT2 receptor expression
Kidneys were harvested from 4-week-old rats, which had been killed by dislocation of the neck (control n = 5 from five litters, LP n = 5 from five litters), for Western blot analysis of AT1 and AT2 receptors. Each kidney was bisected longitudinally, and the medulla removed to leave only the cortex. Membranes were isolated by homogenization and centrifugation, as previously described (Sahajpal & Ashton, 2003). The protein concentration of the resuspended membrane pellet was determined using the Bradford method (Bio-Rad Assay Reagent, Bio-Rad Laboratories, Hercules, CA, USA). Tissue homogenate (50 μg) was then denatured at 80°C for 5 min, and fractionated using an 8% SDS-polyacrylamide gel. Blots were incubated with primary antibody (1:300, affinity-purified rabbit polyclonal AT1 (N-10) or goat polyclonal AT2 (C-18), Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), horseradish-peroxidase-conjugated anti-rabbit secondary antibody (1:5000), and developed with the ECL plus detection kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), as previously described (Sahajpal & Ashton, 2003). Immunoreactivity was quantified by densitometry, and normalized to the control group bands. Within each gel, the mean density of the control group bands was taken to represent 100%; individual densitometry readings for both control and LP bands were then expressed as a percentage of this value, allowing the calculation of standard errors.
, 百拇医药
Glomerular ligand binding assay
Ang II binding was determined in glomeruli isolated from a separate group of 4-week-old animals killed by dislocation of the neck, as previously described (Messenger et al. 1988). Excised kidneys were placed in ice-cold PBS (phosphate-buffered saline, pH 7.4), bisected longitudinally and the medulla removed. The outer cortex was cut into 1 mm cubes, minced and aspirated through a 100 μl pipette tip. The resultant homogenate was washed with 12 ml of ice-cold PBS through graded nylon mesh filters (Cell Micro Sieve, Bio-Design, Inc., New York, USA) of 200 μm and then 100 μm pore size. After centrifugation at 120 g for 5 min at 4°C, the pellet was resuspended in 1 ml of ice-cold PBS, and passed through a 70 μm pore size nylon mesh filter with a further 4 ml of buffer. Glomeruli were recovered at the top of the last sieve by rinsing with PBS. The suspension was centrifuged again at 120 g for 5 min at 4°C, resuspended in PBS, and stored at –80°C. The final suspension was >80% pure and contained isolated, intact glomeruli.
, http://www.100md.com
Saturable binding was assessed using 125I-labelled Ang II (Amersham Pharmacia Biotech) at concentrations from 10–10 to 10–7 M (control n = 9 from six litters, LP n = 7 from six litters). Selective displacement of 125I-Ang II from AT1 and AT2 receptors was demonstrated in a separate series of binding assays by coincubation with losartan (10–4 M; Merck Sharp & Dohme Ltd, Hoddesdon, Herts, UK; control n = 7 from five litters, LP n = 7 from five litters), PD123319 (10–4 M; Sigma, Poole, Dorset, UK; control n = 7 from five litters, LP n = 7 from five litters), or a combination of losartan (10–4 M) and PD123319 (10–4 M) (control n = 4 from four litters, LP n = 4 from four litters). Incubations were carried out in duplicate at room temperature in bovine-serum-albumin-coated tubes. The incubation medium was made up to a total volume of 200 μl consisting of PBS (pH 7.4), the glomerular suspension (equivalent to 20 μg protein) and 125I-labelled Ang II. Non-specific binding was determined by the amount of 125I-labelled Ang II bound in the presence of excess unlabelled Ang II (5 x 10–5 M). Total activity was also determined for each 125I-labelled Ang II concentration. After incubation for 45 min at room temperature, samples were centrifuged at 10 000 g for 10 min at room temperature, resuspended in 1 ml PBS and centrifuged again. Bound activity was determined using a Cobra II gamma counter (Packard, Pangbourne, Berkshire, UK). Specific binding is expressed as femtomoles of bound Ang II per milligram of glomerular protein. The binding affinity constant (KD) and binding capacity (Bmax) were derived by Scatchard transformation of binding data (GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, CA, USA).
, 百拇医药
Renal renin radioimmunoassay
Kidneys were collected from control (n = 9 from five litters) and LP rats (n = 7 from five litters), which had been killed by dislocation of the neck. Tissue was homogenized on ice in Tris/HCl buffer (pH 7.4, 1 ml buffer per 0.1 g tissue) containing enzymatic inhibitors (1% Triton X-100, 2 mM EDTA, 1 μg ml–1 leupeptin, 1 μg ml–1 aprotinin, 0.1 μg ml–1 phenylmethylsulphonyl fluoride and 0.1 μg ml–1 bacitracin), and centrifuged at 1000 g for 20 min at 4°C. Samples were diluted in 0.5 M phosphate buffer (pH 6.5) containing 8.8 mM EDTA, 3 mM 8-hydroxyquinoline sulphate and 5 mM 2,3-dimercaptopropanol to a final concentration of 1:4000 using a two-step dilution; in the second step 100 μl of renin-free plasma was added. Renal renin activity was measured using a REN-CT2 kit (CIS (UK) Limited, High Wycombe, Buckinghamshire, UK), as previously described (Gouldsborough et al. 2003). The kit includes an angiotensin converting enzyme inhibitor solution (specific details of its formulation are not provided by the manufacturer) to prevent degradation of angiotensin I (Ang I) during incubation of samples. Renin activity is expressed in micrograms of Ang I per gram kidney weight per hour.
, 百拇医药
Renal Ang II radioimmunoassay
Kidneys were collected from control (n = 5 from five litters) and LP rats (n = 5 from five litters), which had been killed by dislocation of the neck. A 75 μl aliquot of homogenized kidney, prepared as above, was added to 25 μl assay buffer containing captopril (5 ng mg–1 of tissue) to inhibit Ang II generation in vitro. Ang II was measured by radioimmunoassay, as previously described (Ashton & Balment, 1991), using 3-[125I]iodotyrosyl4 Ang II (5-isoleucine) (2000 Ci mmol–1) and rabbit Ang II antibody (Amersham Pharmacia Biotech). Tissue Ang II concentration is expressed in picomoles per gram kidney weight.
, 百拇医药
Plasma aldosterone radioimmunoassay
Blood from control (n = 5 from five litters) and LP rats (n = 5 from 5 litters), which had been killed by stunning followed by decapitation, was collected into ice-cold heparinized tubes. The plasma was separated by centrifugation, and assayed for aldosterone using a commercial radioimmunoassay kit (Coat-a-Count, Diagnostic Products Corporation, Caernarfon, Gwynedd, UK). The intra-assay coefficient of variation was 3.2%.
, 百拇医药
Statistical analysis
All data are presented as means ± S.E.M. Statistical analysis was by Student's unpaired t test or one-way ANOVA and Duncan's test, as appropriate, with significance ascribed at the 5% level (SPSS for Windows, version 11.5.0, SPSS UK Ltd, Surrey, UK). Saturation curves, binding constants (Bmax) and binding affinity constants (KD) were calculated using GraphPad Prism (version 3.02 for Windows, GraphPad Software). Best fit for one- versus two-site binding equations was compared by F test. The simpler equation was accepted when P > 0.05.
, 百拇医药
Results
Western blot analysis of the kidney cortex from control and LP rats yielded a single band at the expected weight of 45 kDa, corresponding to the Ang II AT1 receptor (Fig. 1). Incubation in the presence of excess immunizing peptide completely blocked antibody binding. AT1 expression in the cortex of 4-week-old LP rats was 62% greater (P < 0.05) than that in control rats. Western blot analysis of the AT2 receptor yielded a band at the expected weight of 45 kDa (Fig. 2). In contrast to the level of AT1 expression, cortical Ang II AT2 receptor expression was significantly lower (P < 0.05) by 35% in LP rats.
, 百拇医药
A, typical immunoblot for the AT1 receptor in the renal cortex of 4-week-old control (C) and low-protein (LP) rats. B, renal cortical AT1 receptor expression in 4-week-old control (open bars, n = 5 from 5 litters) and LP rats (filled bars, n = 5 from 5 litters). Data normalized to the control bands and presented as means ± S.E.M. Statistical comparisons were by Student's unpaired t test. *P < 0.05 control versus LP.
A, typical immunoblot for the AT2 receptor in the renal cortex of 4-week-old control and LP rats. B, renal cortical AT2 receptor expression in 4-week-old control (open bars, n = 5 from 5 litters) and LP rats (filled bars, n = 5 from 5 litters). Data normalized to the control bands and presented as means ± S.E.M. Statistical comparisons were by Student's unpaired t test. *P < 0.05 control versus LP.
, 百拇医药
Figure 3 shows saturation curves of 125I-Ang II binding to glomeruli from LP and control rats in the presence of either the AT1 receptor antagonist losartan or the AT2 receptor antagonist PD123319. One- and two-binding site equations were applied to the saturation curves for 125I-Ang II binding alone, and the goodness of fit compared. For both the control and LP rat binding curves, the one-site model provided the best fit (one versus two-site: control F2,3 = 3.734 P = 0.153; LP F2,3 = 0.976 P = 0.471), therefore the Bmax and KD data have been calculated on this basis. The increase in LP rat AT1 receptor expression detected by Western blotting was reflected by a significant increase in 125I-Ang II binding by the isolated glomeruli. Bmax for LP rats was significantly higher (P < 0.01) than that for control rats (Table 2). KD tended to be lower in LP rats, but this did not reach statistical significance (P = 0.059).
, 百拇医药
A, saturation curves of 125I-Ang II binding to glomeruli isolated from 4-week-old control and LP rats. Binding curves are shown for 125I-Ang II alone (, control n = 9 from 6 litters, , LP n = 7 from 6 litters), 125I-Ang II and losartan (, control n = 7 from 5 litters, , LP n = 7 from 5 litters) and 125I-Ang II and PD123319 ( , control n = 7 from 5 litters, , LP n = 7 from 5 litters). B, Scatchard plots for 125I-Ang II binding alone. Correlation coefficients for the Scatchard plots were significant for both control (r = –0.771, P < 0.05) and LP rats (r = –0.916, P < 0.01).
, 百拇医药
Co-incubating glomeruli with 125I-Ang II and losartan significantly reduced (P < 0.001), but did not completely abolish, 125I-Ang II binding (Fig. 3). Losartan reduced the Bmax of LP rats by 94%, compared with an 85% reduction in control rats (Table 2). Net losartan-sensitive binding in LP rats amounted to 419.4 ± 15.6 compared with 247.2 ± 16.6 fmol (mg glomerular protein)–1 in control animals (P < 0.05). KD values remained similar to those observed for 125I-Ang II alone, and did not differ between LP and control rats. Co-incubation of glomeruli with 125I-Ang II and PD123319 also significantly reduced (P < 0.05) Bmax in both control and LP rats, but to a lesser extent than that seen with losartan. PD12319-sensitive binding was equivalent to 74.2 ± 21.9 and 75.2 ± 23.6 fmol (mg glomerular protein)–1 in control and LP rats, respectively. KD values remained similar to those observed for 125I-Ang II alone; the KD of control rats was significantly higher than that of LP rats (P < 0.05, Table 2). 125I-Ang II binding was only completely abolished by coincubation with losartan and PD123319 (counts not significantly different from background at 10–10–10–7 M Ang II).
, http://www.100md.com
Renal renin activity (Fig. 4A, P = 0.051), renal tissue Ang II (Fig. 4B, P = 0.97) and plasma aldosterone concentrations (Fig. 4C, P = 0.18) did not differ between control and LP rats.
A, renal renin activity (micrograms of angiotensin I per gram kidney weight per hour) in kidneys from 4-week-old control (open bars, n = 9 from 5 litters) and LP (filled bars, n = 7 from 5 litters) rats. B, renal Ang II concentrations (picomoles per gram kidney weight) in kidneys from 4-week-old control (open bars, n = 5 from 5 litters) and LP (filled bars, n = 5 from 5 litters) rats. C, plasma aldosterone concentrations (picomoles per litre) in 4-week-old control (open bars, n = 5 from 5 litters) and LP (filled bars, n = 5 from 5 litters) rats. Statistical comparisons were by Student's unpaired t test.
, 百拇医药
Discussion
This study set out to test the hypothesis that exposure to a maternal LP diet in utero increases expression of glomerular AT1 receptors, and reduces AT2 receptor expression in the young rat. This would account for our previous observations of enhanced glomerular sensitivity to Ang II in LP rats in vivo (Sahajpal & Ashton, 2003), which may contribute to the increase in blood pressure seen in these rats. It provides evidence of an increase in renal AT1 receptor expression and, for the first time, a specific increase in losartan-sensitive Ang II binding capacity in the glomeruli of these rats. These observations are consistent with a recent report (Vehaskari et al. 2004) showing increased AT1 protein and AT1A mRNA expression at 4 weeks of age in a similar, but not identical, model of maternal protein restriction. Furthermore, the increase in AT1 receptor expression observed herein was coupled with a reduction in AT2 receptor expression, which is consistent with a recent report of reduced renal AT2 mRNA in LP rats of a similar age (McMullen et al. 2004). In contrast to earlier observations in neonatal LP rats (Woods et al. 2001), renal renin activity and tissue Ang II concentrations were not lower in LP rats at 4 weeks of age. These data provide evidence that the renin–angiotensin system is not suppressed in the LP rat at this stage of development.
, 百拇医药
There is some published information on the effect of a maternal LP diet on offspring renal renin–angiotensin system activity in the rat, but this is confused by the use of different dietary protein restriction protocols. Renal tissue renin, renin mRNA and Ang II concentrations have been reported to be reduced in LP rats at postnatal days 1 and 5 (Woods et al. 2001). Plasma renin activity has been shown to be reduced in LP rats at 4 weeks of age (Vehaskari et al. 2001), but return to normal at 13 weeks (Langley-Evans & Jackson, 1995). Plasma angiotensin converting enzyme (ACE) activity is increased at 4 weeks, but lung and renal ACE activity are not different (Langley-Evans & Jackson, 1995; Nwagwu et al. 2000). However, apart from our earlier study (Sahajpal & Ashton, 2003) and the recent report by Vehaskari et al. (2004), renal AT1 receptor expression has not been described in LP rats.
, 百拇医药
The renin–angiotensin system plays a critical role in renal development (Guron & Friberg, 2000). This is reflected by the widespread distribution of renin and both AT1 and AT2 receptors within the fetal and neonatal kidney (Gomez et al. 1993). Immunohistochemical analysis has shown that renin immunoreactivity is widespread in the neonatal kidney, being located along the entire length of the afferent arteriole and interlobar artery (Gomez et al. 1989). This contrasts with the adult pattern of distribution in which renin-secreting cells are restricted to the juxtaglomerular position (Gomez et al. 1989). Consistent with its wider distribution in the neonatal kidney, renin mRNA and renal tissue renin activity are also greater in the neonatal kidney by comparison with adult rats (Gomez et al. 1989). In LP rats, renal renin activity is very low in the newborn, relative to controls (Woods et al. 2001), but by 4 weeks of age renin activity and renal Ang II concentrations are comparable with control animals. Similar observations have been reported in another model of maternal protein restriction. Plasma renin activity was low at birth, but gradually increased to exceed that of controls in adulthood (Manning & Vehaskari, 2001). Plasma and renal Ang I and Ang II concentrations were similar (Vehaskari et al. 2004) whereas plasma aldosterone concentrations were elevated (Vehaskari et al. 2001) in LP rats compared with controls at 4 weeks of age. These observations are in broad agreement with our own: renal renin activity, renal Ang II and plasma aldosterone concentrations were all similar in 4-week-old LP and control rats. Taken together, these data suggest that the renin–angiotensin system is no longer suppressed in the LP rat at this stage of development.
, 百拇医药
Renal AT1 receptors first appear in mesenchymal cells and differentiating glomeruli at embryonic day E15 in the rat (Shanmugam et al. 1994). AT1 mRNA increases fivefold from E17 to postnatal day 5 (Tufro-McReddie et al. 1993b), reaching a plateau at around postnatal day 10–20 (Aguilera et al. 1994). AT1 receptors are located throughout the nephrogenic cortex of newborn kidneys, as well as in glomeruli, tubules and vessels (Tufro-McReddie et al. 1993b), the AT1A subtype being the predominant form (Shanmugam et al. 1994). As the kidney matures, the adult pattern of receptor distribution appears, which comprises the glomeruli, proximal tubule, thick ascending limb, distal tubule and collecting duct, as well as the afferent and efferent arterioles, interlobar and arcuate arteries and the descending vasa recta (Miyata et al. 1999). Hence renal AT1 receptor expression in the adult kidney is located primarily in the cortex. The ligand-binding data described herein demonstrate for the first time that 125I-Ang II binding is enhanced in glomeruli from LP rats. Losartan-sensitive binding was 70% higher in LP rat glomeruli, which is consistent with the 62% increase in AT1 receptor expression detected by Western blotting of the renal cortex. These data are also in agreement with previously reported increases in both AT1 receptor protein and mRNA in whole kidney homogenates (Sahajpal & Ashton, 2003; Vehaskari et al. 2004). Therefore, the data presented in the current study are consistent with an increase in AT1 receptor expression in the renal cortex of LP rats, with a specific increase in glomerular receptors. An increase in proximal tubular AT1 receptor expression is unlikely, as Ang-II-mediated Na+ reabsorption did not differ in vivo between LP and control rats (Sahajpal & Ashton, 2003), but we cannot rule out the possibility that AT1 receptors are upregulated in other parts of the nephron or the renal vasculature.
, 百拇医药
In contrast to the AT1 receptor, renal AT2 receptor expression is maximal during nephrogenesis and declines once the kidney matures (Miyata et al. 1999). In the immature kidney, AT2 receptors are located in the nephrogenic zone of the metanephric cortex and ureteric bud (Aguilera et al. 1994). In adult rats, AT2 receptors have been localized to the proximal tubule, collecting duct, arcuate arteries and afferent arterioles (Miyata et al. 1999). There are conflicting reports over AT2 receptor expression in glomeruli (Ozono et al. 1997; Miyata et al. 1999), but functional studies suggest that AT2 receptors can influence the glomerular filtration rate through a vasodilator action (Siragy & Carey, 1997). In the current study, coincubation of glomeruli with 125I-Ang II and PD123319 (AT2 antagonist) significantly reduced the Bmax of both control and LP rats by 17 and 25%, respectively. Furthermore, Ang II binding could not be totally blocked by losartan (AT1 antagonist); a combination of losartan and PD123319 was required to completely block Ang II binding. This suggests that there are low levels of AT2 expression in the glomeruli of both groups. Taking the ligand binding data together with the reduction in AT2 receptor expression observed herein by Western blotting and the reported reduction in AT2 mRNA (McMullen et al. 2004), these observations suggest that the balance between vasoconstriction and vasodilatation is tipped towards constriction in the young LP rat. This is consistent with our earlier observation of a greater reduction in GFR in response to a nonpressor dose of Ang II in the LP rat (Sahajpal & Ashton, 2003).
, 百拇医药
The underlying mechanisms responsible for the increase in AT1 and a decrease in AT2 expression in 4-week-old LP rats are not clear. Blood pressure is already elevated at this age (Sahajpal & Ashton, 2003), which should suppress AT1 expression and renin activity (Tufro-McReddie et al. 1993a). Newborn LP rats have low renal renin activity (Woods et al. 2001), which might be expected to increase glomerular AT1 receptor expression (Wilkes et al. 1988). However, by 4 weeks of age, renal renin activity was comparable between control and LP rats, suggesting that regulation of the renin–angiotensin system changes as the LP rat matures. Increased exposure to maternal glucocorticoids, as a result of placental 11hydroxysteroid dehydrogenase type 2 suppression, has been linked to programming of the renin–angiotensin system in the LP rat (Langley-Evans et al. 1999; Woods, 2000). Clearly, further study is required before the mechanisms involved are fully understood.
, 百拇医药
In summary, this study has confirmed the hypothesis that maternal protein restriction in the rat results in an increase in AT1 receptor expression, coupled with a reduction in AT2 receptors, in male offspring at 4 weeks of age. This was associated with renal tissue renin activity, tissue Ang II and plasma aldosterone concentrations comparable with control animals. The mechanisms responsible for this programming action on the renal renin–angiotensin system are not yet clear, but may be linked to exposure of the fetus to maternal glucocorticoids. The consequence of increased AT1 receptor expression in the glomeruli of LP rats is an increased sensitivity to Ang II, resulting in an inappropriate reduction in GFR. This may be exacerbated by the reduction in nephron number reported in these animals (Sahajpal & Ashton, 2003), leading to a further reduction in the overall filtration capacity of the LP rat kidney, retention of salt and water and eventually an increase in blood pressure.
, 百拇医药
References
Aguilera G, Kapur S, Feuillan P, Sunar-Akbasak B & Bathia AJ (1994). Developmental changes in angiotensin II receptor subtypes and AT1 receptor mRNA in rat kidney. Kidney Int 46, 973–979.
Ardaillou R, Chansel D, Chatziantoniou C & Dussaule JC (1999). Mesangial AT1 receptors: expression, signaling, and regulation. J Am Soc Nephrol 10 (Suppl. 11), S40–S46.
Ashton N & Balment RJ (1991). Sexual dimorphism in renal function and hormonal status of New Zealand genetically hypertensive rats. Acta Endocrinol 124, 91–97.
, http://www.100md.com
Clarke HE, Coates ME, Eva JK, Ford DJ, Milner CK, O'Donoghue PN, Scott PP & Ward RJ (1977). Dietary standards for laboratory animals: report of the Laboratory Animals Centre Diets Advisory Committee. Lab Anim 11, 1–28.
Gomez RA, Lynch KR, Sturgill BC, Elwood JP, Chevalier RL, Carey RM & Peach MJ (1989). Distribution of renin mRNA and its protein in the developing kidney. Am J Physiol Renal Physiol 257, F850–858.
Gomez RA, Tufro-McReddie A, Everett AD & Pentz ES (1993). Ontogeny of renin and AT1 receptor in the rat. Pediatr Nephrol 7, 635–638.
, 百拇医药
Gouldsborough I, Lindop GB & Ashton N (2003). Renal renin–angiotensin system activity in naturally reared and cross-fostered spontaneously hypertensive rats. Am J Hypertens 16, 864–869.
Guron G & Friberg P (2000). An intact renin–angiotensin system is a prerequisite for normal renal development. J Hypertens 18, 123–137.
Helou CM, Imbert-Teboul M, Doucet A, Rajerison R, Chollet C, Alhenc-Gelas F & Marchetti J (2003). Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney. Am J Physiol Renal Physiol 285, F507–514.
, 百拇医药
Khan IY, Taylor PD, Dekou V, Seed PT, Lakasing L, Graham D, Dominiczak AF, Hanson MA & Poston L (2003). Gender-linked hypertension in offspring of lard-fed pregnant rats. Hypertension 41, 168–175.
Langley SC & Jackson AA (1994). Increased systolic pressure in adult rats induced by fetal exposure to maternal low protein diet. Clin Sci 86, 217–222.
Langley-Evans SC (1997). Intrauterine programming of hypertension by glucocorticoids. Life Sci 60, 1213–1221.
, 百拇医药
Langley-Evans SC, Gardner DS & Jackson AA (1996a). Maternal protein restriction influences the programming of the rat hypothalamic–pituitary–adrenal axis. J Nutr 126, 1578–1585.
Langley-Evans SC & Jackson AA (1995). Captopril normalises systolic blood pressure in rats with hypertension induced by fetal exposure to maternal low protein diets. Comp Biochem Physiol A Physiol 110, 223–228.
Langley-Evans SC, Sherman RC, Welham SJ, Nwagwu MO, Gardner DS & Jackson AA (1999). Intrauterine programming of hypertension: the role of the renin–angiotensin system. Biochem Soc Trans 27, 88–93.
, 百拇医药
Langley-Evans SC, Welham SJ, Sherman RC & Jackson AA (1996b). Weanling rats exposed to maternal low-protein diets during discrete periods of gestation exhibit differing severity of hypertension. Clin Sci 91, 607–615.
Lewis RM, Forhead AJ, Petry CJ, Ozanne SE & Hales CN (2002). Long-term programming of blood pressure by maternal dietary iron restriction in the rat. Br J Nutr 88, 283–290.
Manning J & Vehaskari VM (2001). Low birth weight-associated adult hypertension in the rat. Pediatr Nephrol 16, 417–422.
, 百拇医药
McMullen S, Gardner DS & Langley-Evans SC (2004). Prenatal programming of angiotensin II type 2 receptor expression in the rat. Br J Nutr 91, 133–140.
Messenger EA, Stonier C & Aber GM (1988). Differences in glomerular binding and response to angiotensin II between normotensive and spontaneously hypertensive rats. Clin Sci 75, 191–196.
Miyata N, Park F, Li XF & Cowley AW Jr (1999). Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney. Am J Physiol Renal Physiol 277, F437–446.
, 百拇医药
Nwagwu MO, Cook A & Langley-Evans SC (2000). Evidence of progressive deterioration of renal function in rats exposed to a maternal low-protein diet in utero. Br J Nutr 83, 79–85.
Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM & Carey RM (1997). Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension 30, 1238–1246.
Ruan X, Wagner C, Chatziantoniou C, Kurtz A & Arendshorst WJ (1997). Regulation of angiotensin II receptor AT1 subtypes in renal afferent arterioles during chronic changes in sodium diet. J Clin Invest 99, 1072–1081.
, 百拇医药
Sahajpal V & Ashton N (2003). Renal function and angiotensin AT1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci 104, 607–614.
Shanmugam S, Corvol P & Gasc JM (1994). Ontogeny of the two angiotensin II type 1 receptor subtypes in rats. Am J Physiol Endocrinol Metab 267, E828–836.
Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P & Gasc JM (1995). Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland. Am J Physiol Renal Physiol 268, F922–930.
, http://www.100md.com
Sherman RC & Langley-Evans SC (1998). Early administration of angiotensin-converting enzyme inhibitor captopril, prevents the development of hypertension programmed by intrauterine exposure to a maternal low-protein diet in the rat. Clin Sci 94, 373–381.
Sherman RC & Langley-Evans SC (2000). Antihypertensive treatment in early postnatal life modulates prenatal dietary influences upon blood pressure in the rat. Clin Sci 98, 269–275.
, 百拇医药
Siragy HM & Carey RM (1997). The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest 100, 264–269.
Tufro-McReddie A, Chevalier RL, Everett AD & Gomez RA (1993a). Decreased perfusion pressure modulates renin and ANG II type 1 receptor gene expression in the rat kidney. Am J Physiol Regul Integr Comp Physiol 264, R696–702.
Tufro-McReddie A, Harrison JK, Everett AD & Gomez RA (1993b). Ontogeny of type 1 angiotensin II receptor gene expression in the rat. J Clin Invest 91, 530–537.
, 百拇医药
Vehaskari VM, Aviles DH & Manning J (2001). Prenatal programming of adult hypertension in the rat. Kidney Int 59, 238–245.
Vehaskari VM, Stewart T, Lafont D, Soyez C, Seth D & Manning J (2004). Kidney angiotensin and angiotensin receptor expression in prenatally programmed hypertension. Am J Physiol Renal Physiol 287, F262–267.
Wilkes BM, Pion I, Sollott S, Michaels S & Kiesel G (1988). Intrarenal renin–angiotensin system modulates glomerular angiotensin receptors in the rat. Am J Physiol Renal Physiol 254, F345–350.
Woods LL (2000). Fetal origins of adult hypertension: a renal mechanism Curr Opin Nephrol Hypertens 9, 419–425.
Woods LL, Ingelfinger JR, Nyengaard JR & Rasch R (2001). Maternal protein restriction suppresses the newborn renin–angiotensin system and programs adult hypertension in rats. Pediatr Res 49, 460–467., 百拇医药(Vandana Sahajpal and Nick)