Abstract We previously demonstrated that type 1A angiotensin II (Ang II) receptor (AT1A) is the predominant renal subtype and is upregulated by a low sodium diet. We have now tested the hypothesis that upregulation of AT1A mRNA induced by sodium deficiency is renal specific and is mediated by activation of type 1 Ang II receptor (AT1). Male Wistar rats were divided into four groups (n=5 each) and treated for 2 weeks with normal sodium diet (0.5%), normal sodium plus 3 mg/kg per day losartan, low sodium diet (0.07%), or low sodium diet plus losartan. At the end of the 2 weeks, body weight and mean arterial pressure were not different among the four groups (P>.05). Plasma renin activity was elevated by losartan treatment, sodium restriction, or the combination of the two versus control (P<.05). Northern blot analysis showed that the ratio of renal AT1A to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was increased by losartan treatment, sodium restriction, or the combination of the two versus control (P<.05). In contrast, the ratio of adrenal AT1A to GAPDH mRNA was increased only by sodium restriction versus three other groups (P<.05). Thus, sodium deficiency increases AT1A mRNA in both kidney and adrenal gland, while Ang II receptor blockade by losartan prevents low sodium–induced AT1A mRNA only in adrenal gland. We conclude that the increase in AT1A mRNA induced by sodium deficiency is not renal specific and that different mechanisms of regulation exist, ie, in adrenal gland, low sodium–induced upregulation of AT1A is dependent upon Ang II activation of the AT1 receptor, while in kidney, a non-AT1–dependent mechanism is operant.
Knowledge of the mechanisms that regulate Ang II receptor gene expression in the kidney is important for our basic understanding of renal function under normal and pathophysiological conditions because Ang II has direct and indirect actions on the kidney to regulate BP, electrolyte balance, and extracellular fluid volume through binding to specific receptors. With the use of a competitive in vitro autoradiographic ligand binding method, it has been shown that AT1 is the principal renal subtype.1 2 3 Recently, we found that AT1A mRNA in the kidney is plentiful and can be detected readily by Northern blot while the other AT1 receptor subtype, AT1B, cannot be detected by Northern blot, suggesting that AT1A is the predominant renal subtype of the AT1 receptor.4 Similarly, the specific quantification of AT1A receptor mRNA using reverse transcription and PCR amplification showed that the highest absolute AT1A receptor mRNA levels were found in the kidney,5 indicating that AT1A receptors may account for most physiological consequences of Ang II binding.
An important result of our previous work was that AT1A mRNA levels in the kidney are significantly increased by low dietary sodium intake,4 suggesting that expression of the AT1A receptor in the kidney is linked with salt and water homeostasis. Interestingly, sodium depletion, known to activate the renin-angiotensin system, also increases AT1A mRNA levels in the adrenal gland.5 In contrast, adrenal AT1A mRNA levels were not altered in two-kidney, one clip hypertensive rats in which circulating PRA was also elevated.5 These experiments raise questions regarding the mechanism by which low dietary sodium regulates AT1A mRNA levels in both the kidney and adrenal gland. The present study was therefore designed to test the hypothesis that sodium deficiency–induced modulation of AT1A mRNA in both the kidney and adrenal gland is mediated by activation of the AT1 receptor.
Seven-week-old male Wistar rats (Charles River Laboratories, Inc, Wilmington, Mass) were randomly divided into four groups (n=5 in each group) and treated for 2 weeks with normal sodium diet (0.5%, NS group), NS+losartan (DuP group), low sodium diet (0.07%, LS group), or LS+DuP group. The rat food was purchased from Harlan Teklad Diets. DuP (3 mg/kg per day) was dissolved in 0.5 to 1 mL water and given by oral gavage. This dose of DuP was chosen because it has previously been demonstrated to provide Ang II antagonism without lowering BP (personal communication, Dr Pancras C. Wong, 1994, DuPont Merck Pharmaceutical Co). An equal amount of water was given by oral gavage to both the NS and LS rats. 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 2-week diet treatments, all the rats were anesthetized with a single injection of 100 mg/kg IP thiopental sodium. The right carotid artery was catheterized for the measurement of MAP with a Statham 231D pressure transducer (Gould Inc) coupled to a Gould 2400S recorder (Gould Inc). The rats were heparinized (1 U/g), and samples of plasma (3 mL) were collected from the carotid artery and placed in chilled EDTA tubes. PRA was determined using a commercially available radioimmunoassay kit for angiotensin I (Incstar Co).
To obtain tissue for Northern blot analysis, a midline incision in the abdomen was performed, and the left kidney and both adrenal glands were promptly removed, weighed, frozen in liquid nitrogen, and processed for RNA extraction.
Preparation of AT1A cDNA probes has been described in our recent publication.4 Briefly, to make AT1A-specific cDNA probes, the PCR method was used to amplify AT1A cDNA (a generous gift of Dr Tadashi Inagami, Vanderbilt University School of Medicine, Nashville).6 The primers used were 5′-sense primer=5′-TGGCTTACGACCAAAGGACCA-3′ (+1142 to +1162), 3′-antisense primer=5′-CAAAGGGAGACTGATGAGATTG-3′ (+1355 to +1377). These primers span a noncoding region where AT1A and AT1B cDNAs exhibit a minimum sequence homology. 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 PCR product was verified by using a USB sequencing kit (United States Biochemical Co). A 235-bp PCR product was inserted into 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. The AT1A cDNA probes were labeled with [32P]dCTP using a Multiprime DNA labeling system (Amersham Co) to a specific activity of 3×108 CPM/μg. The labeled probes were separated from unincorporated nucleotide using Mini-Spin G-50 DNA purification spin columns (Worthington Biochemical, Co).
RNA Extraction and Northern Blots
Total RNA of kidney and adrenal gland was extracted using the guanidine thiocyanate–phenol–chloroform extraction protocol.7 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% sodium dodecyl sulfate, and 200 μg/mL of 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 it was washed successively in 2×, 1×, and 0.5× SSC (two times, 10 minutes each) containing 0.1% sodium dodecyl sulfate. Stringencies of washes were 65°C. Blots were exposed to XAR-5 x-ray film (Eastman Kodak Co) with intensifying screen. To correct 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 [32P]-labeled GAPDH cDNA probes. Autoradiographic signals were scanned with a laser densitometer (Ultrascan XL, Pharmacia). Relative gene expression was factored for GAPDH mRNA.
Results were expressed as mean±SEM. The data were analyzed by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. Differences were considered statistically significant at the P<.05 level.
There were no significant changes in body weight or MAP among the four groups at the end of the experiment (Table⇓). The ability of low salt intake or DuP treatment to modulate the renin-angiotensin system was confirmed by observation of elevated PRA in the three treatment groups compared with the control group (P<.05, Fig 1⇓).
Renal AT1A mRNA content was determined by Northern blot analysis in each of the four groups of rats (Fig 2A⇓). Blots were then stripped and rehybridized to GAPDH cDNA probes. Densitometric analysis (Fig 2B⇓) indicated that the ratio of AT1A mRNA to GAPDH mRNA was significantly elevated by DuP treatment (1.17±0.10), sodium restriction (1.12±0.03), or the combination of the two (0.96±0.06) compared with controls (0.66±0.04, P<.05).
Fig 3A⇓ shows adrenal AT1A mRNA levels determined by Northern blot analysis in each of the four groups of rats. Blots were then stripped and rehybridized to GAPDH cDNA probes. Densitometric analysis (Fig 3B⇓) indicated that the ratio of AT1A mRNA to GAPDH mRNA was significantly increased by sodium restriction (1.58±0.09, P<.05) compared with controls (0.58±0.05), DuP treatment (0.68±0.03), and the combination of the two (0.69±0.09).
We recently demonstrated that AT1A is the predominant renal subtype of the AT1 receptor, and its mRNA levels in the kidney are significantly increased by low dietary sodium intake.4 The present study was designed to test the hypothesis that upregulation of AT1A mRNA induced by sodium deficiency is renal specific and is mediated by an Ang II–dependent mechanism. We found that blockade of the AT1 receptor with DuP significantly increases AT1A mRNA levels in the kidney but not adrenal gland. In contrast, low dietary sodium significantly increases AT1A mRNA in both kidney and adrenal gland, confirming the prior report that low sodium–induced upregulation of AT1A is not renal specific. Blockade of the AT1 receptor with DuP prevents the low sodium–induced upregulation of AT1A mRNA in adrenal gland but not in kidney, indicating that different mechanisms of regulation of AT1A by sodium are present in the kidney and adrenal gland.
It has been demonstrated that 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.1 2 3 Recently, 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, and 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 be detected only with the more sensitive in situ hybridization.4 Our findings are supported by that of Llorens-Cortes et al,5 who used reverse transcription and PCR amplification and showed that, if expressed as a percentage of total AT1A+AT1B receptor mRNA content, AT1A receptor mRNA content was 73% in the kidney.5 Although no pharmacological approaches are available for distinguishing between AT1A and AT1B, the predominant distribution of AT1A mRNA in the kidney suggests that most physiological consequences of Ang II binding may be mediated by the AT1A receptor subtype. A cautionary note: all of this work is based on the study of message levels and not receptor protein number. Since there are examples of high levels of protein with relatively rare message copy, one cannot rule out the possibility that AT1B receptors are present in the kidney.
Consistent with our previous finding,4 low sodium intake upregulates AT1A receptor mRNA expression. Similarly, sodium depletion upregulates the AT1A receptor mRNA expression in the adrenal gland, suggesting that low sodium–induced upregulation of AT1A is not kidney specific. The mechanism for this upregulation is unclear. Given our understanding of the renin-angiotensin system, increased PRA induced by sodium depletion should result in elevated circulating and/or local Ang II levels. Thus, Ang II could modulate AT1A gene expression by a positive feedback mechanism through the activation of the AT1 receptor. Indeed, this mechanism seems to account for low sodium–induced upregulation of AT1A mRNA in the adrenal gland because DuP fully blocked the response. In contrast, renal AT1A mRNA levels in rats fed a low sodium diet remained elevated in the presence of DuP. There are several potential explanations. One is that DuP has a direct effect on certain specific renal cells to enhance AT1A mRNA expression. In support of this, we dissected the kidney into the cortex and medulla and found that, while low sodium intake selectively enhances AT1 mRNA expression in the medulla, DuP significantly increases AT1 mRNA expression in the cortex only (unpublished data). The second possibility is that, in contrast with the adrenal gland, there is a very low abundance of the AT2 receptor in the kidney. It is possible that excess Ang II induced by DuP treatment binds to the AT2 receptor and has an as yet unknown effect on the regulation of the AT1A gene in the kidney and adrenal gland. This hypothesis could be tested by the simultaneous blockade of the AT1 and AT2 receptors. A third possibility is that elevated Ang II may have negative feedback regulation on AT1A gene expression in the kidney8 but not the adrenal gland, and blockade of the AT1 receptor by DuP eliminates a negative feedback signal and increases renal AT1A mRNA expression. This possibility could be tested by giving Ang II directly to the rats. A fourth consideration is that previous studies on the genomic structure and the promoter region sequence of AT1A have shown that several glucocorticoid-responsive elements exist in the 5′-regulatory region and account for stimulation by aldosterone.9 10 It is possible, therefore, that the release of aldosterone induced by low sodium intake causes the increase in AT1A mRNA expression in both kidney and adrenal gland. Because it has been demonstrated that aldosterone release is AT1 dependent,11 the differences in AT1A mRNA levels between the kidney and adrenal gland after DuP treatment may reflect different responses of the adrenal and kidney tissues to the altered aldosterone concentration. This mechanism could be tested by preventing the release of aldosterone during sodium depletion.
In conclusion, we have demonstrated that sodium depletion increases AT1A mRNA levels in both kidney and adrenal gland. Different mechanisms of regulation of AT1A by sodium exist in these tissues. While low sodium–induced upregulation of AT1A in the adrenal gland is AT1 dependent, a non-AT1–dependent mechanism is operant in the kidney. We propose that the existence of distinct regulatory mechanisms will provide an opportunity for selective manipulation of Ang II receptors in different tissues. This could provide a means for differentially altering ion homeostasis, BP, or tissue growth.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1||=||type 1 Ang II receptor|
|AT1A||=||type 1A Ang II receptor|
|AT1B||=||type 1B Ang II receptor|
|GAPDH||=||glyceraldehyde 3-phosphate dehydrogenase|
|MAP||=||mean arterial pressure|
|PCR||=||polymerase chain reaction|
|PRA||=||plasma renin activity|
This study was supported in part by National Institutes of Health grant HL-52279 and a grant from Merck Research Laboratories to Dr Wang. We thank Dr Tadashi Inagami for the generous supply of AT1A cDNA and Dr Richard D. Bukoski for his critical review of this manuscript.
- Received June 18, 1995.
- Revision received August 18, 1995.
- Accepted September 10, 1995.
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