Differential Regulation of Angiotensin II Receptor Subtypes in Rat Kidney by Low Dietary Sodium
Abstract This study was designed to determine whether expression of renal messenger RNA (mRNA) encoding the two known angiotensin II type 1 (AT1 ) receptor subtypes (AT1A and AT1B) can be regulated by dietary sodium. Seven-week-old male Wistar rats were fed a low-sodium diet (0.07%, n=9) or a normal-sodium diet (0.5%, n=9 [control]) for 14 days. A rat AT1 complementary DNA (cDNA) probe, which hybridizes to mRNA encoding both the AT1A and AT1B receptor subtypes, and cDNA probes, which are selective for AT1A or AT1B mRNA, were used in Northern blot or in situ hybridization analysis. By use of Northern blot analysis, renal mRNA levels for the AT1 and AT1A receptors in rats fed a low-sodium diet were found to be increased twofold (P<.05) compared with control. Because renal AT1B mRNA content was not detected by Northern blot analysis, quantitative image analysis of in situ hybridization with a digoxigenin-labeled cRNA probe made from AT1B cDNA was used. In situ hybridization analysis indicated that AT1B mRNA was expressed in the proximal and collecting tubules of the kidney in rats fed a normal-sodium diet. The low-sodium diet significantly decreased the percent positive staining area of AT1B mRNA in the renal cortex (5.51±0.77% versus 2.73±0.35%, P<.05) and medulla (4.76±0.70% versus 2.01±0.43%, P<.05) compared with the control diet. These results indicate that the increase in AT1 mRNA levels in the kidney induced by low sodium intake is the result of a selective increase in AT1A mRNA and suggest that AT1A is the predominant receptor subtype of AT1 in the kidney. The data also suggest that dietary sodium differentially modulates the expression of genes encoding AT1 receptor subtypes, because there is an inverse relationship between the expression of the AT1A and AT1B subtypes in response to a low-sodium diet. The functional implications are discussed.
- receptors, angiotensin
- sodium, dietary
- gene expression regulation
- blotting, Northern
- in situ hybridization
The renin-angiotensin system plays an important role in blood pressure regulation, electrolyte balance, and extracellular fluid volume control. The effects of angiotensin II (Ang II) are mediated by its binding to specific receptors. These receptors can be divided into at least two different receptor subtypes on the basis of their affinities to synthetic ligands. AT1 receptors are characterized by their high affinity for the nonpeptide Ang II antagonist losartan, whereas AT2 receptors have a high affinity for PD 123177 or CGP 42112A.1 2 With the use of a competitive in situ autoradiographic and ligand binding method, it has been shown that Ang II receptors in the adult kidney are predominantly of the AT1 type.3 4 5
Two AT1 receptor subtypes, AT1A and AT1B, have been cloned and sequenced in the rat.6 7 8 9 10 These two receptor subtypes share 91% identity of the nucleotide sequence in the coding regions but exhibit only 60% identity in their 5′ and 3′ untranslated regions.7 8 9 10 Although AT1A and AT1B share a high degree (96%) of amino acid homology,8 10 they may be functionally different. It has been reported that AT1B has a threefold to fivefold greater affinity for Ang II agonists and antagonists than does AT1A,9 and there are dose-related differences between AT1A and AT1B during Ang II–induced Ca2+ signaling in studies of AT1A and AT1B receptors transfected into COS-7 cells.9 Although it has been noted that AT1 receptor subtypes in the rat brain can be differentially regulated by hormones or changes in dietary sodium,10 11 gene regulation of the AT1 receptor subtypes in the kidney has not been fully investigated. Because of the key role of the kidney in the control of fluid homeostasis and blood pressure, it is important to understand the regulation of these two distinct receptor genes in the kidney. Such studies could lead to further insights into the relevance of these receptor genes in hypertensive disease states and possibly lead to the development of new and more specific therapies. In this study, we used Northern blot and in situ hybridization analysis to test the hypothesis that dietary sodium intake differentially regulates gene expression of the AT1A and AT1B receptor subtypes in the kidney.
Seven-week-old male Wistar rats weighing between 150 and 200 g (Charles River Laboratories, Inc) were randomly divided into two groups and pair-fed a low-sodium (0.07%, n=9) or a normal-sodium (0.5%, n=9) diet (Harlan Teklad Diets) for 14 days. All animal procedures were in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.
Plasma Renin Activity
At the end of the 14 days, all rats were anesthetized with a single intraperitoneal injection of 100 mg/kg thiopental sodium. The right carotid artery was catheterized for the measurement of mean arterial pressure with a Statham 231D pressure transducer (Gould Inc) coupled to a Gould 2400S recorder. The rats were heparinized (1 U/g), and 3-mL samples of plasma were collected from the carotid artery and placed in chilled EDTA tubes. Plasma renin activity was determined using a commercially available radioimmunoassay kit for Ang I (Incstar Co).
Urinary Sodium Concentration
To confirm the effectiveness of dietary sodium restriction, urine samples were collected from the bladder. Urinary sodium concentration was determined using the Synchon EL-ISE sodium chemistry method.12
To obtain tissue for Northern blot analysis, a midline incision was made in the abdomen, and the left kidney was removed, weighed, and frozen in liquid nitrogen and processed for RNA extraction. To obtain tissue for in situ hybridization, the carotid artery was perfused for several minutes at the baseline mean arterial pressure of each rat with saline containing 58.7 g/L polyvinylpyrrolidone and 0.1 mmol/L each of adenosine, verap- amil, and sodium nitroprusside, followed by perfusion fixation with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. The perfusion-fixed right kidney was then harvested for the in situ hybridization studies.
cDNA Probes for Northern Blot Analysis
To make AT1 cDNA probes, a clone pUC19 containing 2.3 kb of the rat AT1A receptor (a generous gift from Dr Tadashi Inagami, Vanderbilt University, Nashville)6 was digested with Kpn I and EcoRI to obtain a 790-bp fragment (−180 to +610; Fig 1⇓). Because this fragment contains the AT1A coding region where AT1A and AT1B cDNAs exhibit high nucleotide sequence identity, this fragment was used as a template for making AT1 cDNA probes. To make AT1A-specific cDNA probes, the polymerase chain reaction (PCR) method was used to amplify AT1A cDNA.6 The 5′-sense primer was 5′-TGGCTTACGACCAAAGGACCA-3′ (+1142 to +1162), and the 3′-antisense primer was 5′-CAAAGGGAGACTGATGAGATTG-3′ (+1355 to +1377). These primers span a noncoding region where AT1A and AT1B cDNAs exhibit a minimum sequence homology (Fig 1⇓). The reaction profile included 30 cycles of denaturation at 95°C for 45 seconds, annealing at 55°C for 60 seconds, and polymerization at 72°C for 90 seconds. The DNA sequence of the PCR product was verified by use of a USB sequencing kit (United States Biochemical Co). A 235-bp PCR product was inserted into the EcoRV site of pBluescript KS(+) vector, and the fragment that was cleaved with EcoRI and HindIII was used as a template for making AT1A cDNA probes. A clone pBluescript KS(+) containing 2.3 kb of the rat AT1B receptor was digested with HindIII and EcoRI to obtain a noncoding region fragment (395 bp, +1246 to +1641, Fig 1⇓).8 This fragment was used as a template for making AT1B cDNA probes. All AT1, AT1A, and AT1B cDNA probes were labeled with [32P]deoxycytidine triphosphate by use of a Multiprime DNA labeling system (Amersham Co) to a specific activity of 3×108 cpm/μg. The labeled probes were separated from unincorporated nucleotides by use of a Mini-Spin G-50 DNA purification spin column (Worthington Biochemical Co).
cRNA Probes for In Situ Hybridization
A 395-bp AT1B cDNA template, described above, was subcloned into the HindIII and EcoRI site of pGEM-3Z. Transcription from the T7 promoter (digested with HindIII) yielded the antisense probe. Transcription from the SP6 promoter (digested with EcoRI) yielded the sense probe. Digoxigenin-labeled cRNA probes were synthesized using the Genius 4 kit (Boehringer Mannheim Biochemicals).
RNA Extraction and Northern Blots
Total kidney RNA was extracted using the guanidine thiocyanate–phenol–chloroform extraction protocol.13 Electrophoresis of 30 μg denatured RNA from each preparation was carried out in a 1% agarose gel containing 2.2 mol/L formaldehyde. RNA was transferred to a positively charged nylon membrane (Fisher Co). The membrane was baked at 80°C for 2 hours in a vacuum oven (Fisher Co) and prehybridized for 5 hours at 42°C in hybridization buffer (50% deionized formamide, 5× Denhardt’s solution, 5× SSC, 0.5% SDS, and 200 μg/mL denatured salmon sperm DNA). The membrane was hybridized with the 32P-labeled probes in the hybridization buffer for 18 to 20 hours at 42°C, then was washed successively in 2×, 1×, and 0.5× SSC containing 0.1% SDS at 65°C. Autoradiograms were made using XAR-5 x-ray film (Eastman Kodak Co). To control the differences in RNA loading, Northern blots were incubated at 90°C for 10 minutes in 20 mmol/L Tris-HCl (pH 8.0) to strip off the cDNA probes and rehybridized with a 32P-labeled 18S rRNA probe. Autoradiographic signals were scanned with a laser densitometer (Ultrascan XL Laser Densitometer). The results are expressed as the ratio of AT mRNA to 18S rRNA.
In Situ Hybridization
In situ hybridizations were carried out as previously described by Johnson et al.14 In brief, the kidney was fixed in 4% paraformaldehyde/0.1 mol/L phosphate buffer, pH 7.4, at 4°C overnight and paraffin embedded. Five-micron–thick tissue sections from the kidney were cut, mounted on superfrost-plus slides (Fisher Scientific), and baked for 1 hour at 55°C. After deparaffinization with xylene and rehydration with graded ethanol, each section was prehybridized in a 100-μL hybridization solution (50% formamide, 4× SSC, 1× Denhardt’s solution, 0.5 mg/mL denatured DNA, 0.25 mg/mL yeast RNA, 10% dextran sulfate) for 1 hour at room temperature and hybridized by addition of 300 ng/mL digoxigenin-labeled AT1B cRNA probes at 55°C overnight. After hybridization, sections were washed with decreasing concentrations of SSC. Immunological detection was accomplished by application of an anti-digoxigenin antibody conjugated to alkaline phosphatase (Genius Nonradioactive Nucleic Acid Detection Kit), followed by calorimetric reaction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate solution. Pilot studies were done in which kidney sections from the control rats were used to determine the proper concentration of the probe and the duration of the color development. To determine background signal, slides were processed after hybridization with digoxigenin-labeled sense cRNA probes or after complete omission of the probe. Kidney sections from the sodium-depleted and control rats were always hybridized at the same time to minimize the intergroup variation.
Quantitative Image Analysis
Quantitative image analysis of the nonradioactive signal was performed using an approach based on previously published methods.15 16 The computerized image-analysis system that was used in the present experiments was bioscan optimas software (Bioscan, Inc) run on a 33-MHz 80486 IBM PC–compatible computer with an Imaging Technology Vision Plus–AT CFG digitalizing card. The inputs for this system included a Nikon Optiphot microscope with a Hitachi HV-C10 CCD color video camera. The outputs from the imaging card were displayed on a 19-inch high-resolution Sony color video monitor. The image-analysis system was calibrated before each section was read, so light level and camera sensitivity were consistent for all measurements. Ten to 15 different 0.39-mm2 areas were circumscribed per section; two sections per rat were measured. The positive staining was automatically marked and converted to optical area units by the computer, and the percent positive staining was calculated using the equation 100%×(positive stained area÷circumscribed area).
Results are expressed as mean±SEM. The data from the normal- and low-sodium groups were analyzed by Student’s unpaired t test. Differences were considered statistically significant at the P<.05 level.
The mean body weight of the rats fed the low-sodium diet (250±4 g) was not significantly different from that of the control rats (253±5 g) at the end of the experiment. Mean arterial pressure was not significantly changed by the sodium diet (125±4 mm Hg) compared with the control diet (116±6 mm Hg). Plasma renin activities in rats fed the low-sodium and the normal diets were 27.0±4.3 ng Ang I · h−1 · mL−1 and 28.8±3.7 ng Ang I · h−1 · mL−1 (P>.05), respectively. Urinary sodium at the end of 14 days was significantly decreased in the low-sodium group (60.8±10.3 mmol) compared with control (218.5±11.6 mmol, P<.01).
Kidney AT1 mRNA and AT1A mRNA contents in the rats consuming the low-sodium and the control diets were determined by Northern blot analysis (Fig 2A⇓ and 2B⇓). Blots were then stripped and rehybridized to 18S rRNA probes. Densitometric analysis indicated that the ratio of AT1 mRNA to 18S rRNA was significantly elevated (Fig 2C⇓) in sodium-restricted rats (1.39±0.25) compared with control rats (0.62±0.21). Equally distinct differences (approximately twofold) in AT1A mRNA gene expression were observed between the rats fed the low-sodium and the normal-sodium diets. The ratio of AT1A mRNA to 18S rRNA was 0.69±0.11 for the control rats versus 1.28±0.11 for the rats consuming the low-sodium diet (P<.05, Fig 2D⇓).
Because no AT1B mRNA was detected by Northern blot analysis, we performed in situ hybridization analysis of AT1B mRNA in the kidneys of the rats fed low-sodium and normal-sodium diets (Fig 3⇓). AT1B mRNA was mainly expressed in the proximal and collecting tubules of the renal cortex, as well as in the collecting tubules of the renal medulla in the rats fed a normal-sodium diet (arrows in Fig 3A⇓ and 3B⇓). Although AT1B mRNA was still detectable after the treatment (arrows in Fig 3C⇓ and 3D⇓), the low-sodium diet markedly attenuated AT1B mRNA expression in both the cortex and the medulla of the kidney (Fig 3C⇓ and 3D⇓). Kidney sections from control rats incubated with the labeled AT1B sense probe (Fig 3E⇓ and 3F⇓) or with the omission of the probe (Fig 3G⇓ and 3H⇓) were devoid of staining. Quantitative image analysis indicated that the low-sodium diet significantly decreased the percent positive-staining area of AT1B mRNA in the renal cortex (2.73±0.35% in rats consuming the low-sodium diet versus 5.51±0.77% in control rats, P<.05) and medulla (2.01±0.43% and 4.76±0.70%, respectively; P<.05, Fig 4⇓).
Although it has been demonstrated that changes in dietary sodium differentially regulate the AT1 receptor subtypes in the rat brain, gene regulation of the AT1 receptor subtypes in the kidney during changes in sodium status has not been studied. The present experiments were therefore designed to test the hypothesis that dietary sodium intake regulates renal mRNA encoding the AT1 receptor subtypes AT1A and AT1B. The major new findings of these studies are that the message for the AT1A subtype is far more abundant than for the AT1B subtype, suggesting that AT1A is the predominant renal subtype of the AT1 receptor and that renal AT1A mRNA levels are significantly increased by low dietary sodium whereas renal AT1B mRNA levels are markedly decreased, suggesting that there is differential regulation of the two receptor subtypes.
Unlike the AT2 receptor, which is abundantly expressed in fetal and adult neuronal tissues, AT1 is the predominant receptor in the rat and human kidney and seems to account for all the known physiological consequences of Ang II binding.3 4 5 Regulation of the AT1 receptor has been demonstrated both in vitro and in vivo. It has been shown that AT1 receptors in primary culture of rat hepatocytes are downregulated by Ang II.17 It has also been shown that AT1 receptor mRNA in the rat kidney is downregulated after aortic coarctation, suggesting that high local Ang II levels, high circulating Ang II levels, or both modulate AT1 receptor gene expression by a negative feedback mechanism.18 In contrast, sodium depletion upregulates the AT1 receptor and its mRNA expression in the adrenal gland,6 19 indicating that circulating Ang II levels may also modulate AT1 gene expression by a positive feedback mechanism. Furthermore, the AT1 receptor and its mRNA in the kidney have been shown to be either decreased19 or unchanged6 by low sodium intake for 4 weeks. Although tissue differences in the regulation of AT1 receptor expression may exist, it is likely that at least some of the inconsistency between these studies is the result of differences in the species studied, the duration and magnitude of the dietary interventions, and the cDNA probes that were used.
A strength of the present study is the fact that we used probes from the 3′-untranslated regions of the cDNAs encoding the AT1A and AT1B receptor subtypes, which allowed us to distinguish between mRNA expression for AT1A and AT1B. Using these probes, we found that AT1A is the predominant subtype of the AT1 receptor in the kidney. This conclusion is based on the fact that AT1A mRNA was readily detected with Northern blot analysis, whereas AT1B could only be detected with the more sensitive in situ hybridization. The results of in situ hybridization indicate that AT1B mRNA is present in the proximal and collecting tubules of the kidney in rats fed a normal diet. The localization of AT1B mRNA appears to be different from that of AT1A. It has been reported that AT1A mRNA localizes in mesangial areas, predominantly at the vascular pole.20 The differential distribution of AT1 receptor subtypes may suggest that AT1A and AT1B play different roles in the kidney under physiological conditions.
Our finding that low sodium intake differentially modulates AT1A and AT1B mRNA expression in the kidney deserves comment. These data suggest that dietary sodium differentially regulates the genes encoding AT1 receptor subtypes, because there is an inverse relationship between the expression of AT1A and AT1B in the kidney in response to a low-sodium diet. If changes in AT1A and AT1B mRNA levels correlate with the final products encoded by these genes, it is likely that AT1A and AT1B receptors play different roles in the adaptation to different sodium intakes. At the present time, however, it is not possible to distinguish between AT1A and AT1B receptors by using currently available methods such as Western blot immunostaining or radioligand binding assays.
Interestingly, an opposite effect of dietary sodium on gene expression of the AT1 receptor subtype in the brain was observed in a study in which it was reported that sodium deprivation enhances the expression of AT1B mRNA and high sodium intake increases the expression of AT1A mRNA.11 Considering that there are differences in the 5′-regulatory regions of the AT1A and AT1B genes,21 22 differential regulation of AT1 receptor subtypes in different tissues in response to sodium may indicate distinct functional specificities.
A question that remains to be answered is how sodium restriction induces changes in mRNA expression of AT1 receptor subtypes. It has been reported that plasma Ang II levels increased 10-fold and 2-fold in rats fed a low-sodium diet, either 0.02% or 0.08%, for 1 week compared with rats fed a 0.3% (normal-sodium) diet.23 24 In the present study, sodium restriction decreased urinary sodium content, but it had no effect on plasma renin activity. Thus, it is likely that the circulating renin-angiotensin system was not activated. One possible explanation is that another compensatory mechanism, such as the release of aldosterone or local renal production of Ang II, was activated in response to the low-sodium diet and contributed to the differential regulation of AT1 receptor subtypes. This possibility deserves additional investigation.
In conclusion, the present studies demonstrate that sodium depletion significantly increases the gene expression of the AT1A receptor subtype and decreases AT1B mRNA levels in the kidney. Such differential regulation of kidney Ang II receptor subtypes by sodium load suggests that they play unique roles in the maintenance of fluid homeostasis. An understanding of the individual roles of these Ang II receptor subtypes in the physiology and pathophysiology of the renin-angiotensin system may provide new information about the regulation of blood pressure and the pathogenesis of arterial hypertension.
This study was supported in part by National Institutes of Health grant HL-52279 (Dr Wang). We thank Dr Donald J. DiPette, Dr Richard D. Bukoski, and Dr Sunil J. Wimalawansa for their critical review of this manuscript and Wilma Frye for her expert secretarial skills.
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