Physiol. Genomics Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 35: 330-340, 2008. First published September 16, 2008; doi:10.1152/physiolgenomics.00187.2007
1094-8341/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Tables
Right arrow All Versions of this Article:
35/3/330    most recent
00187.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ochiai, Y.
Right arrow Articles by Kato, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ochiai, Y.
Right arrow Articles by Kato, N.
Received 14 August 2007; accepted in final form 14 September 2008.
Physiological Genomics 35:330-340 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Dynamic changes of the renin-angiotensin and associated systems in the rat after pharmacological and dietary interventions in vivo

Yoshinori Ochiai , Yi-Qiang Liang , Masakuni Serizawa and Norihiro Kato

Department of Gene Diagnostics and Therapeutics, Research Institute, International Medical Center of Japan, Tokyo, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To address the multiplicity of the renin-angiotensin system (RAS) with particular interest in its local, synergistic regulation, we investigate dynamic changes of the RAS and associated systems in response to external stimuli in the rat. We tested influences of the RAS blockade (candesartan and enalapril), diuretics (hydrochlorothiazide), high lipid diet, and salt loading on tissue mRNA level of 12 principal genes. Under the hemodynamic conditions appropriately predetermined, we quantitatively evaluated mRNA level changes with and without each intervention in five organs–the brain, heart, kidney, liver, and adipose tissues–of male rats (n = 5 each). A total of 250 tissues were examined by real-time PCR. Significant changes in mRNA level (P < 0.05) were found in a drug-, diet- and tissue-specific manner. For instance, 29% of genes (14 out of 48 tissues showing detectable mRNA levels) were differentially regulated by candesartan and enalapril, although both drugs reduced blood pressure to similar extents. When the overall interactions among 12 genes were compared between interventions, the RAS and associated systems appeared to change in the opposite direction between candesartan and high lipid diet in the adipose tissue and between candesartan and salt loading in the heart. Enalapril, however, induced unique patterns of perturbation in the local RAS under the corresponding conditions. Thus, this study provides a fundamental picture of gene expression profile in vivo in the RAS and associated systems. In particular, our data highlight differential regulation between candesartan and enalapril, which may reflect the individual pharmacological properties regarding clinical implications.

renin-angiotensin system; mRNA; gene expression; diuretics; diet


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays a key role in the circulatory system. Acting principally through type 1 angiotensin receptors, the RAS constitutes a master regulator of fluid-electrolyte homeostasis. In this line, clinical impacts of this pathway on regulation of blood pressure (BP) have been highlighted by the impressive efficacy of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) in patients with cardiovascular diseases (24). Also, heated arguments have been brought up about the BP-independent vascular protection by blockade of the RAS (13). In fact, recent human studies with ACE inhibitors and ARBs have demonstrated notable clinical benefits including decreases in the incidence of stroke, end-stage renal disease and diabetes, suggesting important new biological mechanisms mediated by the RAS (7).

Two systems have been known to be physiologically associated with the RAS. One system, which forms an extensive cascade regulating fluid and electrolyte balance in combination with the RAS, is the aldosterone biosynthetic pathway and mineralocorticoid action (5). The other system is the kallikrein-kinin system, triggering of which results in the release of vasoactive kinins, i.e., bradykinin (BK)-related peptides (15). Because of its higher affinity for BK than for angiotensin I (ANG I), ACE is also considered as a kininase (kininase II). A large number of molecular approaches have been undertaken to address the multiplicity of the RAS and its interaction with other vasoactive hormones as outlined in Supplementary Fig. S1A (5, 15, 24).1 Given the complex interplay of these pathways, systematic evaluation of dynamic changes in vivo of the RAS together with its associated systems is essential to understand an overall physiological network and control (or feedback) mechanisms against external stimuli such as pharmacological and dietary interventions. It is known that changes at the level of plasma concentration do not necessarily correlate with changes at the mRNA level (22). Accordingly, as for a total of 12 genes that constitute key components of the target pathways, we investigate mRNA levels in five organs–the brain, heart, kidney, liver, and adipose tissue–by keeping the concept of local RAS in mind (6).

We designed the current study in rat models of hypertension and end-organ damage to answer the following questions: 1) does administration of ACE inhibitors and ARBs provoke similar changes in gene expression across five tested organs when systemic BP is pharmacologically lowered to an equivalent level, 2) do diuretics (hydrochlorothiazide) induce changes in gene expression similarly to the RAS blockade (by ACE inhibitors and ARBs), and 3) do dietary interventions–high lipid diet and salt loading, which can exaggerate insulin resistance and salt sensitivity, respectively–considerably interfere with the local RAS and/or its associated systems?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
We used the spontaneously hypertensive rat of a Japanese colony (SHR/Izm, which is hereafter referred to as SHR) and its subline, stroke-prone SHR (SHRSP/Izm, which is hereafter referred to as SHRSP) for dietary and pharmacological interventions, respectively. Rats were weaned at 4 wk after birth and were placed on a normal rat chow (SP diet, Funabashi Farm, Japan) for the group undergoing pharmacological intervention (male SHRSP, see details below), on SP diet with free access to 1% NaCl drinking water for the group undergoing salt loading (male SHR, n = 5), and on high-fat-high-cholesterol diet (5% lard, 2% cholesterol, and 0.5% cholic acid) for the group undergoing high lipid diet intervention (male SHR, n = 5).

Apart from the salt loading experiments described below, all systolic BP measurements were performed by the tail-cuff method in this study, as previously described (11), in which three consecutive BP readings were taken and averaged for each session. Direct BP measurements were additionally carried out on male SHR (n = 3 each for SP diet alone and for dietary salt loading) with the use of the Dataquest IV system (Data Sciences International) to evaluate hemodynamic influences of salt loading without exposing the rats to cuff-constraint, as previously described (11). In brief, the rats were operated on under anesthesia at 10 wk of age and were allowed to recover for 14 days. Daytime and nighttime average systolic BPs were then recorded with radiotelemetry from 12 wk to 18 wk of age.

At two points, before and after pharmacological and dietary interventions, blood samples were drawn from the tail vein of the rat in the overnight (16 h) fasting state for the measurement of glucose, insulin, total cholesterol, triglycerides, and free fatty acids. On the last day of individual interventions, an overnight, 24-h urine collection was obtained for the measurement of urinary protein.

The rats were killed under pentobarbital anesthesia (50 mg/kg intraperitoneal infusion), and the organs were excised and immediately frozen at –70°C for subsequent RNA extraction. All rats were laboratory animals and were treated in compliance with institutional regulations. This protocol was approved by the animal ethics committee of the Research Institute, International Medical Center of Japan.

Pharmacological intervention.
We chose to test three classes of antihypertensive drugs that were expected to substantially influence the RAS and associated systems in the BP-independent manner: enalapril from ACE inhibitors, candesartan from ARBs, and hydrochlorothiazide from diuretics.

To see whether we could minimize the differences in BP decrease and the resultant BP-dependent effects among these antihypertensive drugs, we first performed a pilot study and then proceeded to a main study as follows. In the pilot study, we tested a few different doses of each drug on 12-wk-old male SHRSP (n = 2 each) as shown in Fig. 1, by referring to the doses reported in the previous studies that had tested the corresponding drugs. The range of the reported doses was: 2–30 mg·kg–1·day–1 for enalapril, 1–10 mg·kg–1·day–1 for candesartan, and 1–20·kg–1·day–1 for hydrochlorothiazide. The rats were given candesartan and hydrochlorothiazide mixed in drinking water, whereas enalapril was given by the gavage method. Systolic BP was measured by the tail-cuff method on every 3–4 days for 4 wk (between 12 and 16 wk of age).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. Temporal changes in systolic blood pressure (BP) with and without administration of three antihypertensive drugs in stroke-prone spontaneously hypertensive rats (SHRSP). A: the results for pilot studies that aimed at determining an appropriate dose of each drug using 2 male SHRSP are shown. B: the results for main studies (n = 5 each) are shown. As for candesartan and enalapril, systolic BP was significantly (P <0.01) different between the drug-intervention group and the control group at every measurement point after 3 days of administration.

 
Based on the results in this pilot study, we determined an appropriate dose of each drug, either 1) beyond the level of which no further BP lowering effects were attained or 2) which was a lower dose among the ones tested when no apparent dose-dependent BP lowering effects were observed.

Dietary intervention.
The dietary interventions were also performed to investigate whether high lipid diet and salt loading, which could exaggerate insulin resistance and salt sensitivity, respectively, considerably interfere with the local RAS and/or its associated systems. The intervention period was restricted to 4 wk between 12 and 16 wk of age, and thereafter the rats were fed on SP diet again. While a substantial part of predisposition to cardiovascular disease traits is assumed to be shared between SHR and SHRSP, some is relatively pronounced in or unique to either of the rat strains. SHR is characterized by prominent hypertension, as well as conspicuous responses to high lipid diet and a modest degree of salt sensitivity. SHRSP is, on the other hand, characterized by severe hypertension and cardiovascular complications such as stroke, cardiac hypertrophy, and renal dysfunction, and almost all male SHRSP would develop stoke with <1 mo of dietary salt loading. Hence, we chose the individual strains to suit the nature of intervention; that is, SHRSP for pharmacological intervention and SHR for dietary intervention.

Tissue processing and RNA isolation.
We homogenized 1 g each of the individual tissues with a 0.5-inch-diameter generator shaft at speed 22,000 rpm for 60 s in a Polytron homogenizer RT1300 (Kinematica, Littau, Switzerland). Total RNA was extracted by using RNeasy maxi kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The quality of RNA was checked with an RNA 6000 Nano LabChip on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The cDNA was then synthesized from 1 µg of the DNase I-treated total RNA by using Omniscript RT (reverse transcriptase) Kit (Qiagen, Valencia, CA) and subjected to real-time PCR amplification.

Real-time PCR amplification.
The PCR primer sequences for target genes were originally designed. Information about the primer sequences is provided in Supplementary Table S1. Real-time PCR amplifications were performed in a 384-well plate in the ABI 7900 HT Sequence Detection System (PE Applied Biosystems) in a total volume of 10 µl, which included 2 µl of template cDNA plus SYBR green I master mixture and a final concentration of 400 nM each of forward and reverse primers. Each sample was analyzed in duplicate, and PCR amplification was performed as follows: 2 min at 50°C and 10 min at 95°C, followed by a total of 40 temperature cycles (15 s at 95°C and 1 min at 60°C). The numbers of copies of the PCR template in a starting sample were calculated by using the SEQUENCE DETECTOR system ver. 2.2.2 incorporated in the ABI 7900 HT Sequence Detection System.

Gene expression quantification.
To draw the standard curve for quantification of target gene copies, plasmids containing a DNA fragment for each of the target genes were generated by TA cloning and used as an external control, while amplification of the peptidylpropyl isomerase A (Ppia) gene was used as an endogenous control.

The concentrations of purified plasmid were determined in micrograms per optical density unit and were converted to molar units, from which the estimated numbers of gene copies were calculated. Then, serial dilutions (7 steps of dilutions from 101 to 106 gene copies) of the plasmid were used to generate standard curves, which were prepared for both the target and the endogenous control. The equation of the line that best fitted these data was determined by regression analysis. The R2 value was calculated for each data set to evaluate the accuracy of the real-time PCR as a quantification method. Linear regression analysis revealed an R2 value of ≥0.99 in all standard curves. We normalized the amount of target gene transcript in our tested samples by using their Ppia fluorescent signal after real-time PCR amplifications. Gene expression changes were determined by the ratio of normalized quantity in treated groups to those in untreated groups (n = 5 each). We set 10 copies per reaction to be a minimum detectable level in the gene expression quantification by the standard curve method (16).

Statistics.
Unpaired Student t-test was used for statistical evaluation of intergroup differences in BP and physiological parameters (with relation to administered drug dose and pharmacological and dietary interventions) and of changes in tissue mRNA levels (with relation to pharmacological and dietary interventions). Values are expressed as means ± SE unless otherwise indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenotype changes induced by interventions.
Temporal systolic BP changes in SHRSP were shown for individual drugs in the pilot and main studies separately (Fig. 1, A and B). In the cases of candesartan and enalapril, systolic BP was markedly decreased as early as day 3 after starting administration at any tested concentrations compared with the control status (i.e., 0 mg·kg–1·day–1). Since no further BP-lowering effects were attained beyond the relevant concentrations, an appropriate dose was set to be 5 mg·kg–1·day–1 for candesartan and 20 mg·kg–1·day–1 for enalapril. In the case of hydrochlorothiazide, on the other hand, there were no apparent dose-dependent decreases in systolic BP throughout the administration period. Therefore, an appropriate dose was set to be 20 mg·kg–1·day–1 for hydrochlorothiazide between two doses tested.

When salt loading was conducted in SHR, increases in daytime BP (which was evaluated by radiotelemetry) were observed in the latter half of the intervention (between 14 and 16 wk of age) (Fig. 2). The BP increases lasted for several days after the salt loading period. Also, when high lipid diet was given to SHR, significant increases in total cholesterol levels were observed compared with the rats fed on normal rat chow (Table 1). Thus, two types of dietary interventions were considered to induce noticeable hemodynamic and biochemical changes in SHR at 16 wk of age, when tissue mRNA levels were investigated.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Time-course changes in systolic BP during and after salt loading in spontaneously hypertensive rats (SHR). Daytime and nighttime systolic BP (SBP) was recorded with the radiotelemetry in male SHR with and without (n = 3 each) salt loading. Salt loading was performed for 4 wk between 12 and 16 wk of age. Each data point represents daily average daytime systolic BP with the rats housed under controlled conditions (temperature 21°C, 12-h day-night cycle). BP values are expressed as means ± SE. *P < 0.05, normal rat chow vs. salt loading by the unpaired t-test.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Physiological and biochemical parameters after pharmacological and dietary interventions in comparison with control diet

 
To facilitate the reader's understanding of the physiological linkage of gene expression to the treatment of hypertension and dietary intervention, a series of physiological parameters including organ weights and urinary protein is demonstrated in Table 1. Among significant changes observed in the present study, it is of note that the relative weight of heart and peri-testis fat pad was decreased by candesartan and enalapril treatment, and the relative heart weight was increased by salt loading. Although the underlying mechanisms remain unclear, BP was significantly (P <0.001) decreased by high lipid diet in SHR. Based on the BP changes thus attained, we could determine the BP-independent mechanism only between enalapril and candesartan, whereas we could not exclude the BP-dependent mechanism when comparing the results for these two drugs with those for the other experiments.

Baseline tissue mRNA level.
Table 2 shows baseline levels of tissue mRNA of 12 genes involved in the RAS and associated systems that were examined in SHRSP and SHR. There appeared to be marked differences in mRNA level among five organs tested in both rat strains. For example, while Agt was expressed ubiquitously, its mRNA levels in the heart were lowest and <1/400 of those in the liver and adipose tissue. >Ren1 was expressed at detectable levels in the kidney alone. As a whole, mRNA levels of 12 genes tended to be lower in SHR than in SHRSP. In some organs, for example, tissue mRNA levels of Agt, Ace2, and Agtr1a in SHR were 1/10 of those in SHRSP, and the mRNA levels of these three genes and two additional genes, Ren1 and Ace, were further lower in normotensive control, Wistar Kyoto rats, than in SHR (Supplementary Table S2). The reason for these findings, i.e., interstrain diversity, remains unclear but may be accounted for in part by differences in genetic backgrounds (or disposition) and hemodynamic overload; e.g., systolic BP levels in SHRSP are 60–70 mmHg higher than those in SHR at 18 wk of age.


View this table:
[in this window]
[in a new window]

  		Table 2. mRNA expression levels of 12 genes involved in the RAS and its associated systems

 
mRNA level changes induced by the RAS inhibition.
For some genes in a given tissue, e.g., Ren1 in the kidney and Nr3c2 in the brain, two types of the RAS blockade induced similar and significant changes in mRNA level (Fig. 3). Several changes were, however, found to occur in a drug-specific manner. For the readers’ reference, the values of fold changes are displayed hereafter in some instances (see also Table 3).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Systematic investigation of 12 genes expression in 5 principal organs after administration of 3 antihypertensive drugs in SHRSP. In each gene, a total of 15 comparisons (3 drugs multiplied by 5 organs) were made to evaluate changes in mRNA level against drug administration. The results are shown by the ratio of average treated (or drug-administered) and untreated mRNA levels, which were measured by quantitative real-time PCR and normalized for a housekeeping gene, Ppia (see MATERIALS AND METHODS). The absolute mRNA levels are largely categorized into 3 groups: high (≥10,000 copies, colored in red), middle (between 1,000 and 10,000 copies, colored in green), and low (<1,000 copies, colored in blue) expression groups. Fold changes are displayed along the log-scale with a base of 2. *P < 0.05 and **P < 0.01, statistical differences were evaluated by unpaired t-test between treated and untreated groups (n = 5 each).

 

View this table:
[in this window]
[in a new window]

  		Table 3. Fold changes in mRNA expression of 12 genes, classified by the type of tissue origin and intervention

 
In the RAS, candesartan increased Ace mRNA levels in the kidney, whereas it decreased Ace mRNA levels in the heart. Enalapril significantly increased Agt mRNA levels in the heart (2.8-fold), and more apparently in this physiological context, enalapril increased Agtr1a mRNA levels in the brain, kidney, and adipose tissue as well as in the heart (2.9-fold). In the adipose tissue, candesartan decreased both Agtr1a and Agtr2 mRNA levels, which was opposite to the changes induced by enalapril. In the aldosterone biosynthetic pathway, candesartan and enalapril significantly changed Nr3c2 mRNA levels in the kidney and adipose tissue in the opposite directions. In the kallikrein-kinin system, although baseline mRNA levels of the relevant genes were relatively modest, candesartan decreased Bdkrb1 and Bdkrb2 mRNA levels in the adipose tissue and Bdkrb2 mRNA levels in the heart, whereas enalapril increased Bdkrb1 mRNA levels in the heart. Since the degree of BP reduction is comparable between candesartan and enalapril administration (Fig. 1), the observed differences in mRNA level changes seem to be attributable to differences in their pharmacological property.

mRNA level changes induced by diuretics.
Administration of hydrochlorothiazide did not influence BP considerably but was speculated to cause substantial volume depletion, which then could lead to significant elevation of Ren1 mRNA levels in the kidney (2.2-fold) (Fig. 3). This diuretic agent induced modestly significant increases in tissue mRNA level of several genes, such as Ace in the liver, Agtr1a in the heart, and Nr3c2 in the kidney.

mRNA level changes induced by dietary intervention.
Although the overall degree of mRNA level changes was relatively modest, high lipid diet and salt loading induced significant changes in tissue mRNA level of several genes (Fig. 4). Both types of dietary interventions similarly and significantly increased mRNA level of Cma1 in the liver and decreased that of Cma1 in the adipose tissue. Notably, in the RAS, high lipid diet increased mRNA level of Agt in 4 organs (between 1.2- and 1.4-fold), Ace in the brain and liver, and both Ace2 and Agtr1a in the adipose tissue, while it decreased mRNA level of Cma1 in the kidney. Salt loading, on the other hand, increased mRNA level of Ace in the heart and adipose tissue, and both Ace2 and Agtr1a in the heart, while it considerably decreased mRNA level of Ren1 in the kidney (0.2-fold).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 4. Systematic investigation of 12 genes expression in 5 principal organs after dietary interventions in SHR. In each gene, a total of 10 comparisons (2 interventions multiplied by 5 organs) were made to evaluate changes in mRNA level against dietary interventions. Details of graph display are same as the legend to Fig. 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has provided direct evidence for dynamic and complex regulations of the RAS and associated systems in the body. Two features of our results are of particular note. First, in view of BP-independent mechanisms, two major pharmacological interventions in the RAS, i.e., administration of ACE inhibitors and ARBs, have been shown to induce partly similar but substantially different patterns of mRNA level changes in a tissue-specific manner. Second, identical interventions, whether pharmacological or dietary, have been proven to induce a variety of mRNA level changes, sometimes in inverse directions, of the same gene among five organs tested.

Several studies in vivo have so far addressed the impact of two major types of the RAS blockade on mRNA level of selected genes (9, 22). While both types of drugs are assumed to suppress ANG II action, the strongest vasoactive compound in the RAS, via Agtr1, some differences in pharmacological properties have been reported to exist between the drugs (2, 25). First, administration of ARBs elevates plasma levels of ANG II, which may result in the activation of Agtr2 contrarily to the direct blockade of Agtr1. Also, administration of ACE inhibitors leads to the concomitant suppression of kininase activity in the kallikrein-kinin pathway as a dual pharmacological property. Moreover, the presence of "aldosterone breakthrough" has been reported for ARBs (19). These differences have been known mostly with relation to systemic changes, i.e., plasma levels, of the corresponding components in the RAS and associated systems. Our systematic investigation in vivo has revealed that enalapril but not candesartan induces significant increases in the Agtr1a and Nr3c2 mRNA levels in several organs. When the overall interactions among 12 genes were compared between interventions, the RAS and associated systems appeared to change in the opposite direction between candesartan and high lipid diet in the adipose tissue and between candesartan and salt loading in the heart. This may indicate the potential, counteractive function of candesartan against metabolic disturbances in a tissue-specific manner. Enalapril, however, induced unique patterns of perturbation in the local RAS under the corresponding conditions (Fig. 5).


Figure 5
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 5. Comparison of the overall interaction among 12 genes in the heart and adipose tissues. Representative results were shown for comparison of the overall interaction among 12 genes tested. Gene symbols with significant (P < 0.05) changes in mRNA level are highlighted in blue (decrease) and in red (increase), and those showing not statistically significant but |fold change| ≥ 1.2 are boxed in blue (decrease) and in red (increase) based on the results for quantitative evaluation with and without each intervention in the heart (A) and adipose tissues (B). Gene symbols with undetectable mRNA levels are colored in gray and those showing |fold change| < 1.2 are not colored (refer to Table 3).

 
Some of findings of interdrug inconsistency are conceptually explainable by differences in the inhibiting (or blocking) points in the RAS between enalapril and candesartan. For example, it has to be noted that in the adipose tissue, mRNA level was significantly decreased for six out of 12 genes tested after administration of candesartan, whereas mRNA level was significantly decreased for only one and significantly increased for two out of 12 genes after administration of enalapril. This indicates drug-specific control systems, which may be related to some physiological pathway triggered exclusively by ARBs, for example, for insulin sensitizing/antidiabetic effects (21).

Despite physiological impacts of the RAS and associated systems on fluid-electrolyte homeostasis and fat metabolism, mRNA level in individual tissues appears to be influenced not only by local feedback but also by systemic changes in neuro-humoral factors and other, as yet undetermined mechanisms in vivo. For example, angiotensinogen is highly expressed in the liver and adipose tissues and excreted into the blood stream. However, changes in its mRNA levels in these organs do not necessarily correlate with changes in its plasma levels. That is, apart from thiazide diuretics, plasma levels of angiotensinogen have been reported to be elevated after a series of interventions examined in the present study (Supplementary Fig. S1). Nevertheless, in mRNA level of angiotensinogen, significant increases were found only for high lipid diet in the liver and adipose tissue.

In the present study, the BP-independent mechanism can be determined between enalapril and candesartan, since BP levels were almost equally reduced in the rat by these pharmacological interventions. However, the comparative analysis of the other pairs needs to be carefully interpreted because of the possible BP-dependent mechanism. Hydrochlorothiazide treatment has not induced significant changes in BP, and it has resulted in modest mRNA-level changes of the genes tested. Also, dietary interventions have induced rather modest mRNA-level changes as a whole despite noticeable hemodynamic and biochemical changes in the rat (Fig. 2 and Table 1). Interestingly, apart from the Ren1 gene, salt loading and two types of the RAS blockade and diuretics do not induce mRNA level changes in the inverse direction that could be expected from the viewpoint of fluid-electrolyte homeostasis. Another finding of interest is that, though modest, Agt mRNA level is significantly increased after high lipid diet in four out of five organs, which validates an important role of dietary lipids in the induction of Agt gene expression in vivo (4).

Moreover, our systematic investigation has identified the potential presence of some interactions in the RAS and associated systems. For example, Agtr1a and Nr3c2 tended to respond to administration of enalapril in the same direction (Fig. 3), and Ace2 and Agtr1a tended to respond to dietary interventions in the same direction (Fig. 4). It remains to be defined whether these are chance effects or reflect some unnoticed physiological interplay in vivo.

The detailed mechanisms linking the RAS gene expression profile to the physiological parameters measured in the present study remain to be defined. However, distinct patterns of mRNA level changes induced by a series of pharmacological and dietary interventions allow us to make some speculation in the physiological and molecular relevance of the RAS blockade. For example, in the heart of SHRSP, both candesartan and enalapril significantly reduced heart weight, in accordance with the previous studies reporting the beneficial effects of ACE inhibitors and ARBs on cardiac (or left ventricular) hypertrophy in hypertensive rats (14). Cardiac hypertrophy and fibrosis are characteristic of hypertensive heart disease. ANG II produced locally has been known to be the dominant hormone responsible for these pathologic processes (3). Moreover, increased ACE activity and mRNA expression have been observed in rat hearts with pressure overload (20) and hypertrophic hearts induced by high salt intake (27). This tissue-specific induction of the ACE gene may also contribute to the detrimental roles of the RAS in cardiac hypertrophy, and therefore, the RAS blockade at the point of ACE is considered to be efficacious. In this line, significant reduction in ACE mRNA levels after candesartan treatment (Fig. 3) seems to support its antihypertrophic effects, additionally to the direct blockade of ANG II at the point of Agtr1a. Since major components of the RAS–renin, ANG II, and aldosterone–can exert profibrotic effects on cells, pharmacological inhibition or activation of the local RAS leads to changes in extra-cellular matrix (ECM) turnover, i.e., synthesis and degradation, in the heart (3). Notably, Kim et al. (12) reported that ACE inhibitor (enalapril) and ARB (losartan) could equally reduce cardiac hypertrophy and suppress abnormal expression of ECM genes except for the lack of inhibition of collagen type I expression by enalapril. Likewise, our ongoing microarray analysis indicates that some ECM genes, such as subtypes of collagen and fibronectin, are differentially regulated in the heart when mRNA level changes are compared between candesartan and enalapril treatment (unpublished data). Thus, despite a comparable degree of heart weight decrease, the contents of improvement of the ECM remodeling in cardiac hypertrophy may differ to a certain extent between two types of the RAS blockade.

There are a few limitations in the present study. Above all, even if there are physiological similarities between rats and humans, some components in the RAS have been reported to differ between two species in terms of enzymatic function (8). For example, chymase, the most important enzyme responsible for non-ACE conversion of ANG I to ANG II, shows striking species variation (1). Accordingly, we should carefully interpret experimental findings when we extrapolate the physiological and clinical relevance of rats to humans. Also, we should keep in mind confounding influences of interstrain phenotypic diversity inherent to the rat models tested. That is, some differences in mRNA levels are known to exist between SHR and SHRSP because of interstrain differences in BP levels and genetic makeup (17). This issue is beyond the scope of our present study but needs to be taken into account if we are to attempt to compare the results for pharmacological interventions in SHRSP with those for dietary interventions in SHR by using not the direction of changes (or fold changes) but the absolute values of mRNA level. Furthermore, end-organ protection by the RAS inhibition has been reported in the rat models studied, but it often takes longer (>12 wk) drug administration before clinical effects, e.g., reduction in proteinuria, become manifest (10, 20). This issue requires further examination of time-course changes by focusing on individual end-organ damage. Because the present study primarily aimed at systematizing all the different gene modification observed in several contrasting studies published in this field, we have focused on changes in the mRNA levels but not in the protein levels. This needs to be further examined to verify the physiological and/or clinical relevance of our findings.

In summary, the RAS and associated systems are likely to be regulated in a multiphased manner against a variety of external stimuli; i.e., via intra- and extracellular generation of vasoactive hormones and activation of autocrine and/or paracrine hormones. In view of the concept of local RAS (6), introduced almost 20 years ago, and of prorenin (18), contributing to tissue angiotensin generation, this multiplicity of regulation plays a key role in determining dynamic changes in vivo of the RAS and associated systems. Although findings in the present study remain phenomenological, they provide a fundamental picture of gene expression profile in vivo in the RAS and associated systems. Further extensive studies are warranted to clarify the underlying mechanisms and will then allow us to understand the pathophysiological significance or therapeutic implications of the RAS blockade toward end-organ protection.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Grant-in-Aid from the Ministry of Education, Cultures, Sports, Science and Technology of Japan (#16390229 and #17019068) and a grant for the Core Research for the Evolutional Science and Technology from the Japan Science Technology Agency.


   ACKNOWLEDGMENTS
 
We thank Drs. Makoto Miyagishi, Miyuki Makaya, Naoko Birukawa, and Masato Isono for valuable advice and technical assistance. Enalapril and hydrochlorothiazide were provided by Towa Pharmaceutical Co. Ltd. (Osaka, Japan), and candesartan was provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan).


   FOOTNOTES
 
Address for reprint requests and other correspondence: N. Kato, Dept. of Gene Diagnostics and Therapeutics, Research Inst., International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan (e-mail: nokato{at}ri.imcj.go.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akasu M, Urata H, Kinoshita A, Sasaguri M, Ideishi M, Arakawa K. Differences in tissue angiotensin II-forming pathways by species and organs in vitro. Hypertension 32: 514–520, 1998.[Abstract/Free Full Text]
  2. Bakris GL, Siomos M, Richardson D, Janssen I, Bolton WK, Hebert L, Agarwal R, Catanzaro D. ACE inhibition or angiotensin receptor blockade: impact on potassium in renal failure. VAL-K Study Group. Kidney Int 58: 2084–2092, 2000.[CrossRef][Web of Science][Medline]
  3. Berk BC, Fujiwara K, Lehoux S. ECM remodeling in hypertensive heart disease. J Clin Invest 117: 568–575, 2007.[CrossRef][Web of Science][Medline]
  4. Boustany CM, Bharadwaj K, Daugherty A, Brown DR, Randall DC, Cassis LA. Activation of the systemic and adipose renin-angiotensin system in rats with diet-induced obesity and hypertension. Am J Physiol Regul Integr Comp Physiol 287: R943–R949, 2004.[Abstract/Free Full Text]
  5. Connell JM, Davies E. The new biology of aldosterone. J Endocrinol 186: 1–20, 2005.[Abstract/Free Full Text]
  6. Danser AH. Local renin-angiotensin systems: the unanswered questions. Int J Biochem Cell Biol 35: 759–768, 2003.[CrossRef][Web of Science][Medline]
  7. Dzau V. The cardiovascular continuum and renin-angiotensin-aldosterone system blockade. J Hypertens Suppl 23: S9–S17, 2005.[CrossRef][Medline]
  8. Hollenberg NK. Implications of species difference for clinical investigation: studies on the renin-angiotensin system. Hypertension 35: 150–154, 2000.[Abstract/Free Full Text]
  9. Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension 43: 970–976, 2004.[Abstract/Free Full Text]
  10. Kanazawa M, Kohzuki M, Kurosawa H, Minami N, Ito O, Saito T, Yasujima M, Abe K. Renoprotective effect of angiotensin-converting enzyme inhibitor combined with alpha1-adrenergic antagonist in spontaneously hypertensive rats with renal ablation. Hypertens Res 27: 509–515, 2004.[CrossRef][Web of Science][Medline]
  11. Kato N, Nabika T, Liang YQ, Mashimo T, Inomata H, Watanabe T, Yanai K, Yamori Y, Yazaki Y, Sasazuki T. Isolation of a chromosome 1 region affecting blood pressure and vascular disease traits in the stroke-prone rat model. Hypertension 42: 1191–1197, 2003.[Abstract/Free Full Text]
  12. Kim S, Ohta K, Hamaguchi A, Yukimura T, Miura K, Iwao H. Effects of an AT1 receptor antagonist, an ACE inhibitor and a calcium channel antagonist on cardiac gene expressions in hypertensive rats. Br J Pharmacol 118: 549–556, 1996.[Web of Science][Medline]
  13. Kurtz TW. False claims of blood pressure-independent protection by blockade of the renin angiotensin aldosterone system? Hypertension 41: 193–196, 2003.[Free Full Text]
  14. Lijnen P, Petrov V. Renin-angiotensin system, hypertrophy and gene expression in cardiac myocytes. J Mol Cell Cardiol 31: 949–970, 1999.[CrossRef][Web of Science][Medline]
  15. Moreau ME, Garbacki N, Molinaro G, Brown NJ, Marceau F, Adam A. The kallikrein-kinin system: current and future pharmacological targets. J Pharm Sci 99: 6–38, 2005.[CrossRef][Web of Science]
  16. Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR. Green I monitoring during amplification. Biotechniques 24: 954–958, 1998.[Web of Science][Medline]
  17. Nabika T, Nara Y, Ikeda K, Endo J, Yamori Y. Genetic heterogeneity of the spontaneously hypertensive rat. Hypertension 18: 12–16, 1991.[Abstract/Free Full Text]
  18. Nguyen G. Renin/prorenin receptors. Kidney Int 69: 1503–1506, 2006.[CrossRef][Web of Science][Medline]
  19. Rossi GP. Aldosterone breakthrough during RAS blockade: a role for endothelins and their antagonists? Curr Hypertens Rep 8: 262–268, 2006.[CrossRef][Web of Science][Medline]
  20. Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS, Lorell BH. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation. J Clin Invest 86: 1913–1920, 1990.[Web of Science][Medline]
  21. Schupp M, Janke J, Clasen R, Unger T, Kintscher U. Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation 109: 2054–2057, 2004.[Abstract/Free Full Text]
  22. Sechi LA, Griffin CA, Giacchetti G, Valentin JP, Llorens-Cortes C, Corvol P, Schambelan M. Tissue-specific regulation of type 1 angiotensin II receptor mRNA levels in the rat. Hypertension 28: 403–408, 1996.[Abstract/Free Full Text]
  23. Sironi L, Gelosa P, Guerrini U, Banfi C, Crippa V, Brioschi M, Gianazza E, Nobili E, Gianella A, de Gasparo M, Tremoli E. Anti-inflammatory effects of AT1 receptor blockade provide end-organ protection in stroke-prone rats independently from blood pressure fall. J Pharmacol Exp Ther 311: 989–995, 2004.[Abstract/Free Full Text]
  24. Watanabe T, Barker TA, Berk BC. Angiotensin II and the endothelium: diverse signals and effects. Hypertension 45: 163–169, 2005.[Abstract/Free Full Text]
  25. Weber MA. Hypertension treatment and implications of recent cardiovascular outcome trials. J Hypertens Suppl 24: S37–S44, 2006.[Medline]
  26. Yamamoto K, Mano T, Yoshida J, Sakata Y, Nishikawa N, Nishio M, Ohtani T, Hori M, Miwa T, Masuyama T. ACE inhibitor and angiotensin II type 1 receptor blocker differently regulate ventricular fibrosis in hypertensive diastolic heart failure. J Hypertens 23: 393–400, 2005.[CrossRef][Web of Science][Medline]
  27. Zhao X, White R, Van Huysse J, Leenen FH. Cardiac hypertrophy and cardiac renin-angiotensin system in Dahl rats on high salt intake. J Hypertens 18: 1319–1326, 2000.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Tables
Right arrow All Versions of this Article:
35/3/330    most recent
00187.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ochiai, Y.
Right arrow Articles by Kato, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ochiai, Y.
Right arrow Articles by Kato, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.