Dietary Salt Intake Modulates Angiotensin II Type 1 Receptor Gene Expression
Abstract This study aimed to characterize the influence of dietary salt intake on the gene expression of angiotensin II type 1 (AT1) receptor subtypes in different organs. Male Sprague-Dawley rats were fed low salt (0.2 mg/g), normal salt (6 mg/g), or high salt (40 mg/g) diets for 5, 10, and 20 days. mRNA levels for the two AT1 receptor subtypes were determined in adrenal gland, kidney, liver, and lung. In all of the organs examined, with the exception of the adrenal glands, low salt diet led to a transient decrease in the abundance of AT1A receptor mRNA but not of AT1B mRNA, which reached their nadirs between days 5 and 10 of feeding. In the adrenal gland, in which the AT1B receptor is predominant, low salt diet led to a transient increase in the expression of this receptor gene, with a maximum around day 10 of feeding. High salt diet exerted no significant influence on AT1 receptor gene expression in these organs. These findings indicate that the rate of salt intake, in particular, a reduction of salt intake, significantly influences AT1 receptor gene expression in an organ-, time-, and subtype-dependent fashion. It appears that AT1 receptor subtypes are differentially influenced by low salt intake, in that AT1B receptor gene expression increases and AT1A receptor gene expression decreases in this situation. This differential response of AT1 receptor gene expression may be relevant for the organism to be able to adapt to a reduction in oral salt intake.
The RAAS is essentially involved in the maintenance of sodium homeostasis of an organism. Consequently, RAAS activity is regulated by the rate of salt intake, which inversely determines the rate of renin secretion1 from the kidneys. Renin is the regulatory enzyme for the generation of Ang II, which in turn is the biological key effector of the RAAS. Thus, Ang II increases vascular resistance, increases salt reabsorption in the kidney directly by tubular effects and indirectly by the stimulation of aldosterone production, and increases salt appetite by an action on the brain.2 All of these effects are mediated by AT1 cell surface receptors. Together with the concentration of circulating Ang II, the availability of AT1 receptors therefore essentially determines the efficacy of the RAAS. There is evidence that the rate of salt intake also influences the sensitivity of certain body reactions toward Ang II. Thus, the sensitivity of aldosterone production toward Ang II appears to be enhanced during low salt intake.3 4 5 6 7 In fact, it has been found that the number of AT1 receptor binding sites increases in the adrenal zona glomerulosa8 during low salt intake; evidence has been produced that this is due to the enhancement of AT1 receptor gene expression in this organ,4 6 7 9 suggesting that the rate of salt intake may influence the RAAS not only by regulating renin secretion but also by regulating AT1 receptor gene expression.
Upregulation of AT1 receptor gene expression by low salt diet was recently reported for the whole kidney.10 11 A microdissection study of the kidney provided information that low salt diet causes downregulation of AT1 gene expression in the glomeruli and upregulation of AT1 gene expression in tubular structures.12 For the liver, downregulation of AT1 receptor gene expression by low salt intake was reported,12 whereas for the brain, upregulation of AT1 receptors was reported during high salt diet.4
Taken together, the currently available information about the influence of dietary salt intake on AT1 receptor gene expression produces a rather inconsistent picture. Several explanations exist for these complex findings. First, the rate of salt intake may influence AT1 receptor gene expression in an organ-specific fashion. Second, the apparent divergent findings may have resulted from the different durations of the diets studied, which ranged from 5 to 30 days. Third, the apparently inconsistent findings may be due to technical limitations intrinsic to the techniques applied, which included receptor binding studies, Northern blotting, and PCR.
To resolve these questions and determine the influence of dietary salt on Ang II receptor gene expression, we investigated systematically the time-dependent influence of salt intake on AT1 receptor gene expression in different organs. The AT1 receptor family comprises two subtypes: AT1A and AT1B receptors. We therefore also assessed the influence of the rate of salt intake on the relative expression of these two AT1 receptor subtypes. To this end, rats were fed a low and high salt diet for 5, 10, and 20 days. The abundance of AT1 mRNA as well as the relative proportion of AT1A and AT1B receptors was assayed in adrenal glands, brain, heart, kidneys, liver, and lungs. Our findings indicate that the influence of salt intake on AT1 receptor gene expression is both organ and time dependent.
Male Sprague-Dawley rats (200 to 250 g) obtained from local animal facilities were used for the experiments. Six different groups each with five rats were maintained for 5, 10, or 20 days on rat chow balanced in all respects except for a low salt (0.2 mg/g) or high salt (40 mg/g) content (Altromin). Controls received food with normal salt content (6 mg/g) (Altromin). At the end of the experiments, the rats were killed by decapitation. Blood was sampled from the carotid arteries, centrifuged, and frozen at −20°C. The kidneys, adrenals, livers, lungs, hearts, and brains were quickly removed, weighed, and rapidly frozen in liquid nitrogen. The organs were stored at −80°C until isolation of total RNA, which was extracted from frozen organs as described by Chomczynski and Sacchi.13
RNase Protection Assay for AT1 Receptor mRNA
AT1 receptor mRNA in different organs was measured by RNase protection assay as described previously.14 The precise distinction of AT1A and AT1B receptor subtypes was not possible because of the high homologous amino acid sequence between the receptor subtypes, resulting in cross-hybridizations. The total abundance of AT1 receptor mRNA was determined with the combination of specific AT1A and AT1B cRNAs, which produce protected fragments of the same size. Simultaneous hybridization of RNA with both cRNA probes (the specific activities of AT1A and AT1B are the same) consequently yielded an AT1 signal that was the sum of AT1A and AT1B mRNAs.
Vectors for the generation of the specific AT1A and AT1B cRNA probes were constructed by subcloning a partial cDNA clone of rat AT1A or AT1B mRNA in the transcription vector pSP65 (Promega-Serva). The AT1A clone contains a 298-bp sequence (139 to 437)15 and the AT1B clone a 217-bp fragment (127 to 344).16 Linearization with Sty I and subsequent in vitro transcription with SP6 RNA polymerase (Amersham International) yielded a 98-bp protected cRNA fragment for both AT1 receptor subtypes. Transcripts were labeled with [α-32P]GTP (400 Ci/mmol, Amersham International) and purified on a Sephadex G50 spin column. For hybridization, total RNA was dissolved in a buffer containing 80% formamide, 40 mmol/L piperazine-N,N′-bis(2-ethanesulfonic acid), 400 mmol/L NaCl, and 1 mmol/L EDTA (pH 8). Depending on the AT1 mRNA levels in the different tissues, 30 to 100 μg total RNA was hybridized in a total volume of 50 μL at 60°C for 12 hours with 5×105 cpm radiolabeled AT1A or AT1B probe. RNase digestion with RNase A and T1 was carried out at room temperature for 30 minutes and terminated by incubation with proteinase K for 30 minutes at 37°C (0.1 mg/mL containing 0.4% sodium dodecyl sulfate). The fragments were purified by phenol/chloroform extraction, ethanol precipitation, and subsequent electrophoresis on a denaturing 10% polyacrylamide gel. After autoradiography of the dried gel at −80°C for 1 day, bands representing the combination of protected AT1A and AT1B fragments were excised from the gel, and radioactivity was counted with a liquid scintillation counter (1500 Tri-Carb, Packard Instrument Co).
RNase Protection Assay for GAPDH mRNA
The abundance of rat GAPDH mRNA in total RNA was measured by RNase protection assay as described previously.17 A GAPDH cRNA probe containing a 342-bp fragment of rat GAPDH cDNA18 was generated from a pGEM-4Z vector (Promega-Serva) after linearization with HindIII and transcription with SP6 polymerase. GAPDH mRNA was used as a standard RNA controlling the quality of RNA preparation. Total RNA (1 μg) was hybridized under the conditions described for the determination of AT1A and AT1B mRNAs.
RT-PCR for AT1A, AT1B, and β-Actin mRNAs
AT1A and AT1B
The proportion of AT1A and AT1B mRNAs in different tissues was determined by PCR as described previously.5 Sense (5′-CCAAAGTCACCTGCATCATC-3′) and antisense (5′-CACAATCGCCATAATTATCCTA-3′) primers were designed from the cDNA sequences common to rat AT1A15 and AT1B16 receptors. These primers correspond to regions where no sequence divergence exists between the receptor subtypes. The resulting fragment is 305 bp long (from 723 to 1028 bp in the AT1A sequence and from 630 to 935 bp in the AT1B sequence). The AT1A cDNA fragment but not the AT1B cDNA fragment contains an EcoRI restriction site at position 851 of the sequence.15
For PCR of β-actin, the sense primer (5′-CCGCCCTAGGCACCAGGGTG-3′), which spanned the border of the second exon and second intron, and the antisense primer (5′-GGCTGGGGTGTTGAAGGTCTCAAA-3′), which bound in the fourth exon, were used, resulting in an amplified fragment of 298 bp.19 For PCR reaction, 2 μg total RNA of different tissues was reverse transcribed with 200 U Moloney murine leukemia virus RT (GIBCO-BRL) with the use of standard protocols. To allow the quantification of AT1A, AT1B, or β-actin from one cDNA sample, we used oligo(dT)12-18 (Sigma-Aldrich) for priming the RT reaction (0.5 μg per reaction). From the total volume of 22 μL RT mixture, 3 μL undiluted cDNA for AT1A and AT1B and 3 μL cDNA diluted 1:200 for β-actin were used for PCR and mixed with 1 μL of each primer (10 pmol/μL), 2 μL of 10× PCR buffer, 1 μL dNTP (2.5 mmol/L), and 1 U Taq polymerase (Beckman). The reaction was performed in a final volume of 26 μL.
PCR conditions, depending on the abundance of AT1 mRNA in the different organs, were 30 to 34 cycles consisting of denaturation at 94°C (60 seconds), annealing at 52°C (60 seconds), and extension at 72°C (30 seconds). PCR was completed by a final extension step of 10 minutes at 72°C.
To distinguish between AT1A and AT1B receptor amplification products, we added 1.5 μL EcoRI (25 U/mL) to 20 μL of the PCR product obtained with the AT1 primers. The mixture was incubated at 37°C for at least 60 minutes and the reaction terminated at 94°C for 2 minutes. The digestion yielded fragments of the expected sizes of 128 and 177 bp, as has been visualized by agarose gel electrophoresis and ethidium bromide staining (Fig 1⇓). For quantitative analysis, the density of the bands was assessed within the linear range of UV detection by a densitometer (Bioprofil, Fröbel Labortechnik).
Determination of Plasma Renin Activity
Plasma renin activity was determined with a commercially available radioimmunoassay kit for Ang I (Sorin, Biomedica).
Significance levels were calculated with the Bonferroni test. A value of P<.05 was considered significant.
To assess the efficacy of the low and high salt diets on systemic functions in the rats, we measured plasma renin activity and hematocrit. Low salt diet led to a significant increase of hematocrit and also caused a marked and sustained increase of plasma renin activity, whereas high salt diet continuously suppressed renin secretion (Table⇓).
Abundance of AT1 mRNA in Different Organs During Normal Salt Diet
For the characterization of AT1A and AT1B receptor gene expression in various organs, we examined the presence of AT1 mRNA in the adrenal gland, brain, heart, kidney, liver, and lung by RNase protection. AT1 mRNA was determined in 50-μg aliquots of total RNA from different tissues. As shown in Fig 2⇓, the overall expression levels of AT1 receptor genes differed markedly in the different organs. The abundance of AT1 mRNA as estimated from the RNase protection assay was lung=kidney>adrenal gland>liver≫heart>brain (Fig 3⇓, top). The adrenal gland and liver showed AT1 mRNA levels about 0.5-fold lower than in lung and kidney, whereas in heart and brain, AT1 mRNA abundance fell to 1% to 4% of the values found in lung and kidney.
Distribution of AT1A and AT1B mRNAs in Different Organs During Normal Salt Diet
To determine the proportion of AT1A and AT1B mRNAs in various organs, we used an RT-PCR method that coamplifies both subtypes with the same pair of oligonucleotide primers and distinguishes between the two PCR products by a restriction digest of the AT1A sequence (Fig 1⇑). We found a marked preponderance of AT1A receptor gene expression in liver, lung, heart, and brain, where AT1A mRNA exceeded AT1B mRNA by between 10- and 18-fold (Fig 3⇑, bottom). In the kidney, expression of the two AT1 receptor genes was more balanced, yielding a ratio of AT1A to AT1B of about 3. In the adrenal gland, the expression of the two AT1 receptor genes was reversed, in that AT1B mRNA abundance was about twofold higher than AT1A mRNA abundance (Fig 3⇑, bottom).
Because of the very low abundance of AT1 mRNA in heart and brain, it could not be reliably assayed by RNase protection. Therefore, we focused on the influence of salt intake on AT1 receptor gene expression in adrenal gland, kidney, liver, and lung. To normalize AT1 receptor gene expression in these organs during high and low salt intakes, AT1 mRNA levels were set into proportion to the RNase protection hybridization signal obtained for the housekeeper gene GAPDH, which was assayed in 1-μg aliquots of the total RNA preparations. The GAPDH mRNA signals were organ dependent but were independent of the rate of salt intake.
Influence of Salt Intake on AT1 Receptor Gene Expression in Adrenal Gland
High salt diet (40 mg/g) did not significantly change AT1 mRNA levels in the adrenal gland during 3 weeks of feeding. However, low salt diet (0.2 mg/g), compared with the control diet, led to a significant increase of AT1 mRNA levels, with a maximum around day 10 of feeding when AT1 mRNA was increased by about a factor of 3 (Fig 4⇓, top). With ongoing diet, AT1 mRNA approached control values.
High salt diet did not affect the proportion of AT1A to AT1B receptor gene expression in the adrenal glands (Fig 4⇑, bottom); however, low salt diet significantly lowered the ratio of AT1A to AT1B receptors (Fig 4⇑, bottom).
Calculation of the abundance of AT1 receptor subtype compared with the overall abundance of AT1 mRNA and the proportion of AT1A to AT1B mRNA revealed that AT1B mRNA increased during low salt intake in the adrenal gland, whereas AT1A expression was not clearly changed (Fig 5⇓, bottom).
Influence of Salt Intake on AT1 Receptor Gene Expression in the Kidney
In the kidney, high salt intake had no effect on AT1 receptor gene expression during the 20-day period. However, low salt intake decreased AT1 receptor mRNA levels significantly by about a factor of 1.6 around day 10 of the diet compared with the normal chow diet (Fig 6⇓, top).
The proportion of AT1A to AT1B mRNA in the kidney remained unaffected by high salt diet, whereas low salt intake caused a pronounced decrease of the ratio of AT1A to AT1B (Fig 6⇑, bottom), which, as indicated by the calculated AT1A and AT1B mRNA levels in Fig 5⇑ (top), resulted from a decrease of AT1A mRNA.
Influence of Salt Intake on AT1 Receptor Gene Expression in the Liver
During the 3 weeks of low salt diet, we found a downregulation of AT1 receptor gene expression in the liver around day 5 of feeding (Fig 7⇓, top) that was paralleled by a decrease of the ratio of AT1A to AT1B (Fig 7⇓, bottom). The fall of AT1 mRNA levels resulted from a lowering of AT1A gene expression, as indicated by the calculated values for mRNA subtypes (Fig 8⇓, bottom).
Elevating the NaCl content in the food from 6 to 40 mg/g caused an upregulation of AT1 receptor gene expression in the liver after 20 days of the diet (Fig 7⇑, top), in contrast to results found in the other organs.
Influence of Salt Intake on AT1 Receptor Gene Expression in the Lung
As shown in Fig 9⇓ (top), high salt intake did not change overall AT1 gene expression in the lungcompared with expression in rats kept on a normal salt diet. Also, high salt diet did not change the relative distribution of AT1A and AT1B mRNA (Fig 9⇓, bottom).
Low salt diet led to a transient decrease of AT1 mRNA levels in the lung (Fig 9⇑, top). This decrease of AT1 mRNA was paralleled by a decrease of the ratio of AT1A to AT1B mRNA (Fig 9⇑, bottom). As indicated by the calculated values for AT1A and AT1B mRNA in Fig 8⇑ (top), the decrease of the ratio was caused by lowering of AT1A mRNA.
The RAAS is essentially involved in the sodium homeostasis of mammals. Consequently, the RAAS is controlled by the rate of sodium intake in the sense of negative feedback. Since the biological activity of the RAAS is mediated by AT1 receptors, whether the rate of salt intake exerts its well-known influence not only on the expression20 and secretion1 of renin but also on the expression of AT1 receptors could be of major importance. A few studies have already addressed this question and provided evidence that the rate of oral salt intake in fact influences AT1 receptor gene expression.4 6 7 9 10 11 12 However, the results obtained are partly contradictory and do not allow recognition of a general effect of salt intake on AT1 receptor gene expression. Therefore, we investigated systematically the influence of salt intake on the expression of the two AT1 receptor genes in different organs.
The overall expression levels of AT1 mRNA in the different organs occurred over a broad range, being highest in the lung and lowest in the total brain, in which the abundance of AT1 mRNA was about 1% of the value found in the lung (Fig 3⇑, top). An organ distribution of AT1 receptor gene expression similar to that found in the present study has already been reported.9 21 22
The relative proportion of AT1A and AT1B receptor gene expression also varied among the different organs (Fig 3⇑, bottom). Although the AT1A receptor gene was highly predominant in liver, lung, heart, and total brain, AT1B mRNA was readily detectable in the kidney and predominated in the adrenal gland (Fig 1⇑). Again, this organ distribution of AT1A and AT1B receptor gene expression is in good accordance with previous reports,7 22 23 24 25 26 thus supporting the validity of the assays used in the present study.
Reducing the NaCl content in the chow from 6 to 0.2 mg/g produced similar effects on AT1 mRNA levels in kidney, liver, and lung. We found a temporally transient fall of AT1 mRNA levels that was paralleled by a decrease of the ratio of AT1A to AT1B. Consequently, the fall of AT1 receptor gene expression in these organs appeared to result from an almost selective downregulation of AT1A gene expression, whereas AT1B receptor gene expression remained unchanged or tended to increase (Fig 5⇑, top; Fig 8⇑). A decrease of AT1 gene expression during low salt intake has already been reported for the liver.12 Our findings, however, disagree with the results of Du et al10 and Wang and Du11 reporting an enhancement of AT1A gene expression in the kidney during low salt intake. The situation in the kidney might be more complex, since Cheng et al12 reported a differential response of AT1 receptor gene expression to changes in salt intake in renal glomeruli and renal tubules of the rabbit. In any case, our data suggest that the overall effect of low salt intake on renal AT1A receptor gene expression is inhibitory.
As opposed to the other organs examined in the present study, low salt intake markedly stimulated AT1 receptor gene expression in the adrenal gland, as has already been reported.6 7 9 Our findings now suggest that this effect is due to an enhancement of AT1B receptor gene expression, which increased in a time-dependent fashion, reaching a maximum of expression around day 10 of the diet. Calculation of AT1B abundance from the AT1 mRNA level and the ratio of AT1A to AT1B would also suggest a marginal increase of AT1B mRNA in the adrenal gland after 10 days of a low salt diet (Fig 5⇑, bottom). However, to say that the regulation of the AT1 gene in the adrenal gland behaves in a manner opposite to that of the other organs should be considered with some caution.
Taken together, the findings suggest that low salt intake negatively influences AT1A receptor gene expression and positively influences AT1B receptor gene expression. To our knowledge, low salt intake would be one of the first conditions for a differential regulation of the two AT1 receptor subtypes.
What could be the physiological meaning of a differential response of AT1A and AT1B gene expression to low salt intake? There is little doubt that AT1B receptors mainly mediate the RAAS-dependent stimulation of aldosterone production by low salt intake in the adrenal gland.23 An upregulation of this receptor subtype could be favorable for aldosterone production and consequently for sodium saving during low salt intake. Compatible with this concept is the finding that the sensitivity of aldosterone production toward Ang II increases during low salt diet,3 4 5 6 7 which is surprising because it represents a kind of positive feedback.
The AT1A receptor is mainly expressed in the smooth muscle cells of the vasculature15 23 26 and is therefore relevant for vascular resistance and blood pressure. It is well known that an increase of circulating Ang II usually causes an increase of blood pressure but not if sodium deficiency is the reason for the increase of Ang II levels. Certainly, a number of reasons may contribute to the prevention of hypertension in this situation; one reason could be the downregulation of AT1A receptors in the vasculature. Thus, the differential regulation of AT1A and AT1B receptor gene expression by the rate of salt intake may reflect the capability of the organism to dissociate the sodium-saving and blood pressure effects of the RAAS under certain conditions.
The mechanisms by which low salt intake stimulates AT1B receptor gene expression in the adrenal gland and inhibits AT1A receptor gene expression in other organs cannot be determined from the present study. Future experiments must clarify whether a humoral factor or eventually changes of the extracellular sodium concentration are the primary mediators. A cell culture study has provided preliminary evidence that changes of ionic concentration could influence AT1 receptor gene expression.27 On the other hand, it has been suggested that Ang II is involved in the upregulation of AT1 receptor gene expression in the adrenal gland during low salt intake.23
Although lowering the salt content of the food produced pronounced effects on AT1 gene expression, we found no significant changes of this parameter in adrenal gland, kidney, and lung when the salt content was increased from 6 to 40 mg/g. There was a tendency for AT1A and AT1B mRNA to behave in a manner opposite to the changes seen with a low salt diet; however, these effects were not significant. One could speculate that the sixfold increase in the salt content was not strong enough to elicit significant changes or that the regulatory range for the influence of salt content on AT1 receptor gene expression is less than 6 mg/g.
There are some reports that a high salt intake during 3 weeks of diet led to a significant increase of AT1A receptor gene expression in rat brain.4 This previous report is in accordance with our results found in the liver, where AT1 mRNA levels increased around day 20 of high salt intake. In any case, it should be noted that renin secretion was significantly changed in the rats kept on the high salt diet, suggesting that the sensitivities of AT1 receptor gene expression and of the renin system differ in response to changes in salt intake.4
Another finding obtained in the present study was that the influence of salt intake on AT1 receptor gene expression was temporally transient; that is, diet effects on AT1 mRNA levels appeared in an organ- and subtype-dependent manner within 5 to 10 days of diet, but after 3 weeks of diet, AT1 receptor mRNA levels had returned close to control values. However, the influence of salt intake on renin secretion remained sustained, indicating again that the mechanism by which salt intake changes AT1 receptor gene expression is different from mechanisms by which salt intake influences the renal renin system.
Taken together, the findings obtained in the present study suggest that oral salt intake exerts a systemic influence on AT1 receptor gene expression, in that AT1A receptor gene expression is positively related to the rate of salt intake, whereas AT1B receptor gene expression appears to be inversely related to salt intake. Future research must elucidate the systemic and cellular mechanisms by which salt intake modulates AT1 receptor gene expression.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1B||=||angiotensin type 1, 1a, 1b receptors|
|PCR||=||polymerase chain reaction|
The authors gratefully acknowledge the expert technical assistance provided by Marlies Hamann, Karl-Heinz Götz, and Marie-Luise Schweiger.
- Received June 4, 1996.
- Revision received July 2, 1996.
- Accepted September 25, 1996.
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