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Endocrinology 148(5):2453–2457 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1287
Synergistic Expression of Angiotensin-Converting Enzyme (ACE) and ACE2 in Human Renal Tissue and Confounding Effects of Hypertension on the ACE to ACE2 Ratio Shigeyuki Wakahara, Tadashi Konoshita, Shinichi Mizuno, Makoto Motomura, Chikako Aoyama, Yasukazu Makino, Norihiro Kato, Ichiro Koni, and Isamu Miyamori Third Department of Internal Medicine (S.W., T.K., S.M., M.M., C.A., Y.M., I.M.), Fukui University School of Medicine, Fukui 910-1193, Japan; Department of Gene Diagnostics and Therapeutics (N.K.), Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan; and Molecular Genetics of Cardiovascular Disorders (I.K.), Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8641, Japan Angiotensin-converting enzyme (ACE) 2, a newly emerging component of the renin-angiotensin system, is presumed to be a counterregulator against ACE in generating and degrading angiotensin II. It remains to be elucidated how mRNA levels of these two genes are quantitatively regulated in the kidney and also what kind of clinicopathological characteristics could influence the gene expressions in humans. Seventyeight cases of biopsy-proven renal conditions were examined in detail. Total RNA from a small part of each renal cortical biopsy specimen was reverse transcribed, and the resultant cDNA was amplified for ACE, ACE2, and glyceraldehyde-3phosphate dehydrogenase with a real-time PCR system. Then we investigated the relationship between clinicopathological variables and mRNA levels adjusted for glyceraldehyde-3phosphate dehydrogenase. Statistically significant correlation was not observed between any clinicopathological variables and either of the gene expressions by pairwise comparison. However, a strong correlation was observed between the gene expressions of ACE and those of ACE2. More-
T
HE RENIN-ANGIOTENSIN SYSTEM (RAS) plays important roles in the pathophysiology of cardiovascular and renal conditions (1, 2). Recently the existence of the local RAS in tissues such as heart, kidney, and blood vessel has been a focus of investigation (3). Various studies have reported the pivotal characterizations of the tissue RAS (4 – 6). Angiotensin-converting enzyme (ACE) is one of the most important components in the RAS. From a genetic viewpoint, ACE variants have been tested for their association with cardiovascular and renal diseases, resulting in the controversy (7–13). On the other hand, as a novel component of the RAS, a homolog of ACE, designated as ACE2, has been First Published Online February 15, 2007 Abbreviations: ACE, Angiotensin-converting enzyme; ACEi, ACE inhibitor; Ang 1–7, angiotensin 1–7; Ang II, angiotensin II; ARB, angiotensin receptor blocker; Ccr, creatinine clearance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HT, hypertension; PAC, plasma aldosterone concentration; PRA, plasma renin activity; RAS, reninangiotensin system; s-ACE, serum ACE; UNaE, urinary sodium excretion; UPE, urinary protein excretion. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
over, the ACE to ACE2 ratio was significantly higher in subjects with hypertension (HT) than that in subjects without HT. Whereas parameters of renal function, e.g. urinary protein excretion (UPE) and creatinine clearance (Ccr), are not significantly related to the ACE to ACE2 ratio as a whole, the HT status may reflect disease-induced deterioration of renal function. That is, UPE and Ccr of subjects with HT are significantly different from those without HT, in which a significant correlation is also observed between UPE and Ccr. Finally, stepwise regression analysis further revealed that only the HT status is an independent confounding determinant of the ACE to ACE2 ratio among the variables tested. Our data suggest that ACE2 might play an important role in maintaining a balanced status of local renin-angiotensin system synergistically with ACE by counterregulatory effects confounded by the presence of hypertension. Thus, ACE2 may exert pivotal effects on cardiovascular and renal conditions. (Endocrinology 148: 2453–2457, 2007)
isolated (14, 15). This enzyme is highly expressed in the endothelial cells of heart, kidney, and testis (16) and mainly degrades angiotensin II (Ang II), which acts as a strong vasoconstrictor and is involved in cellular proliferation, to angiotensin 1–7 (Ang 1–7), which is thought to act as a vasodilator and to be involved in apoptosis and growth arrest in contrast to ACE activity of generating Ang II and degrading Ang 1–9. Thus, ACE2 might be a potent counter-regulator against ACE and might play a significant role in the regulation of cardiovascular and renal conditions (17–21). Especially, a recent report suggested that deletion of the ACE2 gene leads to the development of angiotensin II-dependent glomerular injury (22). However, no quantitative, comparative assessment was made for the gene expressions of ACE and ACE2, and hence, it is of interest to characterize the tissue RAS especially in human renal tissue. In this study, we evaluated, first, the relationship between clinicopathological variables and renal tissue expression of the two genes; second, the relationship between ACE and ACE2 gene expressions; and finally, the relationship between clinicopathological variables and the ACE to ACE2 ratio of renal tissue expression in humans. Consequently, real-time
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PCR with a very small part of renal biopsy specimen was applied, making an accurate quantification of mRNA possible, despite the inability in similar protein evaluation because of the limitation of specimens’ quantity. Materials and Methods Subjects and clinicopathological evaluation Subjects were 78 patients (38 male and 40 female subjects) with biopsy-proven renal diseases. The study was approved by the ethics committee of Fukui University (no.17–12), and informed consent for participation was obtained from all individuals. For each subject, salt intake was standardized to 10 g daily in the hospital. Their basal clinical characteristics was as follows (values are means ⫾ sd): age 39.1 ⫾ 19.8 yr, systolic blood pressure 123.2 ⫾ 22.4 mm Hg, diastolic blood pressure 72.9 ⫾ 14.4 mm Hg, urinary sodium excretion (UNaE) 129.2 ⫾ 74.5 mEq/gCr, urinary protein excretion (UPE) 1435 ⫾ 2652 mg/gCr, serum creatinine level 0.87 ⫾ 0.43 mg/dl, creatinine clearance (Ccr) 96.6 ⫾ 52.3 ml/min, plasma renin activity (PRA) 2.4 ⫾ 3.4 ng/ml䡠h, plasma aldosterone concentration (PAC) 100.4 ⫾ 55.9 pg/ml, and serum ACE (sACE) 11.1 ⫾ 4.1 (IU/liter). The numbers of patients administering each class of antihypertensive drugs at the admission for biopsy were as follows: calcium channel blocker, seven; ␣-blocker, three; diuretics, nine; ACE inhibitor (ACEI), one; angiotensin receptor blocker (ARB), one. However, administered ACEIs and ARBs were replaced by calcium channel blocker or ␣-blocker at least 1 wk before biopsy because ACEIs and ARBs are known to have a significant effect on ACE2 gene expression (23). The histological diagnosis of the subjects consisted of seven with minor renal abnormalities, seven with benign nephrosclerosis, 41 with primary glomerulonephritis including four with minimal change nephrotic syndrome, eight with diabetic nephropathy, 12 with lupus nephritis, and three with others. Serum ACE levels were measured in 60 cases by Kasahara’s method (24). After 30 min rest in the supine position, blood samples were drawn for the measurement of PRA and PAC. Hypertension (HT) was defined as blood pressure above 140/90 mm Hg measured in the sitting position on two separate occasions and/or under antihypertensive medication. Glomerular injury was classified into four grades (I-IV) according to the method of Jackson and Johnston (25). Tubulointerstitial injury was also classified into three grades (I-III) according to the method of Bertani et al. (26).
Quantification of mRNA of ACE and ACE2 in human renal tissues A small part of the renal cortex specimen (about 2 mm) was obtained from each subject by echographically guided percutaneous renal biopsy with 18G needle. Each specimen was presumed to contain about 20 –30 glomeruli in this procedure. Immediately after obtaining the biopsy specimen, total RNA was extracted using RNA-Bee (Tel-Test, Inc., Friendswood, TX) according to the protocol recommended for a small amount of samples followed by DNase treatment with deoxyribonuclease (RT grade) (Nippon Gene Co., Ltd., Tokyo, Japan) by the manufacturers’ instructions. Single-strand cDNA was synthesized by a reverse transcriptase reaction with 500 ng/l Oligo-dT (Toyobo Co. Ltd., Osaka, Japan), 1.0 U/l RNase inhibitor (Toyobo), 50 mm dithiothreitol, and Moloney murine leukemia virus reverse transcriptase (Toyobo) and incubated for 1 h at 37 C. The resultant cDNA was amplified for ACE and ACE2 as well as a housekeeping gene, GAPDH. To access genomic DNA contamination, controls without reverse transcriptase were included. Primer sequences were adopted from previous reports for ACE, ACE2 (16) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (27). The primer for ACE were 5⬘-CCGAAATACGTGGAACTCATCAA-3⬘ (sense) and 5⬘-CACGAGTCCCCTGCAT-CTACA-3⬘ (antisense) located from 2430 to 2451 bp (exon 17) and 2475 to 2495 bp (exon 18), respectively (28, 29), and amplifies a 68-bp region of the mRNA. The primer for ACE2 were 5⬘CATTGGAGCAAGTGTTGGATCTT-3⬘ (sense) and 5⬘-GAGCTAATGCATGCCATTCTCA-3⬘ (antisense) located from 2864 to 2886 bp (exon 18) and 2950 to 2971 bp (exon 18), respectively (15), and amplifies a 107-bp region of the mRNA. The primer for GAPDH were 5⬘-CCCATCACCATCTTCCAGGAG-3⬘ (sense) and 5⬘-GTTGTCATGGATGACCTTGCC-3⬘ (anti-
Wakahara et al. • ACE/ACE 2 Expression Ratio in Human Renal Tissue
sense) located from 211 to 231 bp (exon 4/exon5) and 475 to 495 bp (exon 7), respectively (30, 31), and amplifies a 284-bp region of the mRNA. The real-time PCR took place with a final volume of 20 l containing 0.5 mm of forward and reverse primer and 2 l of single-strand cDNA template in 2⫻ QuantiTect SYBR Green PCR master mix (QIAGEN Inc., Tokyo, Japan). The highly specific measurement of mRNA was carried out for ACE, ACE2, and GAPDH using the LightCycler system (Roche Diagnostics Inc., Tokyo, Japan). Each sample was run and analyzed in duplicate. The absolute quantification was performed using the samples of known concentration prepared from amplified DNA fragments extracted and purified from agarose gel for electrophoresis for ACE, ACE2, and GAPDH. With this method, 6 orders linearity was attained in serial dilutions of the sample. The mRNA levels of ACE and ACE2 were adjusted for as the values relative to those of GAPDH, which is the most standard housekeeping gene for mRNA assessment.
Statistical analysis Statistical analyses were performed with SPSS (version 11.0J; SPSS Japan, Inc., Tokyo, Japan). When the distribution of measurement values was significantly deviated, the values were transformed so as to be consistent with the normal distribution before statistical analysis; square root transformation was performed for PRA and logarithmic transformation was performed for UNaE, UPE, and ACE and ACE2 gene expressions (after adjustment with GAPDH). Because the distribution of the [net ACE to net ACE2] ratio was significantly deviated (kurtosis ⫽ 3.42, skewness ⫽ 13.1, Kolmogorov-Smirnov test z ⫽ 2.52, P ⬍ 0.01), we alternatively adopted the ratio of each log-transformed value, i.e. the [log ACE to log ACE2] ratio (kurtosis ⫽ 0.16, skewness ⫽ 1.70, KolmogorovSmirnov test z ⫽ 0.65, P ⫽ 0.80) as the ACE to ACE2 gene expression ratio for statistical analysis. Still, its distribution was platykurtic, but no further transformation was thought to be adequate. First, linear regression analysis was performed to test the correlation between a pair of variables under investigation. One-way ANOVA followed by the twotailed Student t test was then performed for the nominal and ordinal data. Stepwise regression analysis was further performed to evaluate the confounding factors for the ACE to ACE2 gene expression ratio with the entry threshold of test variables set to be P ⫽ 0.05. Data are expressed as means ⫾ sd, unless otherwise indicated.
Results Clinicopathological variables and ACE and ACE2 gene expression
First, the relationship between clinicopathological variables and the tissue ACE and ACE2 gene expressions was assessed (Tables 1 and 2). Although not statistically significant, an interesting tendency of correlation was observed between UPE and ACE2 gene expression (r ⫽ ⫺0.231, P ⫽ 0.051). No significant correlation was, however, observed between any other variables and the two gene expressions (Table 1). No significant difference in the two gene expressions was observed when each TABLE 1. Correlation between clinical variables and renal tissue mRNA levels of ACE and ACE2 ACE
Age (yr) UNaE (mEq/gCr) UPE (mg/gCr) Ccr (ml/min) PRA (ng/ml䡠h) PAC (pg/ml) s-ACE (IU/liter)
ACE2
r
P
r
P
0.11 0.05 ⫺0.04 0.18 ⫺0.11 0.18 0.05
0.36 0.70 0.72 0.12 0.37 0.14 0.71
0.08 0.11 ⫺0.23 0.19 ⫺0.10 0.18 0.04
0.50 0.36 0.051 0.09 0.80 0.13 0.77
To normalize the data distribution for statistical analysis, square root transformation was performed for PRA, and logarithmic transformation was performed for UNaE, UPE, and ACE and ACE2 gene expressions (after adjustment with GAPDH).
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TABLE 2. Clinicopathological variables and renal tissue mRNA levels of ACE and ACE2 Variables (n)
Gender Male (38) Female (40) HT ⫺ (58) ⫹ (20) Glomerular sclerosis scorea I (36) II (29) III and IV (12) Tubulointerstitial scoreb I (25) II (39) III (13)
ACE Mean ⫾
ACE2 Mean ⫾
P
SD
P
SD
2.12 ⫾ 0.65 2.02 ⫾ 0.10
0.48
2.90 ⫾ 0.71 3.04 ⫾ 0.57
0.34
2.04 ⫾ 0.57 2.15 ⫾ 0.62
0.48
3.02 ⫾ 0.61 2.80 ⫾ 0.73
0.19
2.10 ⫾ 0.55 2.05 ⫾ 0.61 1.91 ⫾ 0.49
0.73 (vs. II) 0.48 (vs. III, IV) 0.33 (vs. I)
3.00 ⫾ 0.70 3.01 ⫾ 0.63 2.82 ⫾ 0.50
0.80 (vs. II) 0.40 (vs. III, IV) 0.51 (vs. I)
2.01 ⫾ 0.78 2.10 ⫾ 0.52 2.10 ⫾ 0.31
0.56 (vs. II) 0.95 (vs. III) 0.71 (vs. I)
2.97 ⫾ 0.59 2.95 ⫾ 0.72 3.00 ⫾ 0.16
0.87 (vs. II) 0.81 (vs. III) 0.92 (vs. I)
a
Glomerular injury was classified into four grades (I–IV) according to the method of Jackson and Johnston (25). Tubulointerstitial injury was classified into three grades (I–III) according to the method of Bertani et al. (26). One of the samples was not adequate for glomerular assessment and the other one was not adequate for tubulointerstitial assessment. To normalize the data distribution for statistical analysis, logarithmic transformation was performed for ACE and ACE2 gene expressions (after adjustment with GAPDH). b
of them was tested for association with clinicopathological variables including histological scores (Table 2). Relationship between ACE and ACE2 gene expressions
Next, we evaluated a potential correlation between ACE and ACE2 gene expressions. This relationship is thought to be of interest because the two genes’ products have opposite effects on the generation of Ang II, i.e. the opposite action regarding tissue damage. Our data showed that there was a significant correlation between ACE and ACE2 gene expressions (r ⫽ 0.396, P ⬍ 0.001) (Fig. 1). A similar significant correlation was observed in the glomerulonephritis group (n ⫽ 41, r ⫽ 0.382, P ⬍ 0.014). Clinicopathological aspects and ACE to ACE2 gene expression ratio
Based on this finding of a significant correlation, we further evaluated confounding factors (or perturbation) on the
ACE to ACE2 ratio of human renal tissue expression. No significant correlation was observed between clinical variables and the ACE to ACE2 gene expression ratio (Table 3). Neither did histological scoring for glomeruli and interstitial injury show significant relationship with the ACE to ACE2 gene expression ratio (Table 4). However, as for the HT status, the ACE to ACE2 ratio was significantly higher in subjects with HT than subjects without HT (P ⫽ 0.0496) (Table 4). Stepwise regression analysis also revealed that only the HT status was an independent determinant of the ACE to ACE2 ratio among the clinicopathological variables tested, which explained a total of 5.7% of the variance in this measure (R2 ⫽ 0.057, P ⫽ 0.045). Discussion
The RAS is a coordinated hormonal cascade and a central regulator of cardiovascular, renal, and adrenal functions that regulate fluid and electrolyte balance and arterial pressure (1, 2). Besides, the RAS plays a key role in the pathophysiology of various diseases of the cardiovascular and renal systems (32, 33). ACE removes two carboxyl-terminal amino acids from angiotensin I giving rise to Ang II, which is degraded only by peptidases at different amino acid sites to form fragments in the classical concept for the RAS. Recently an TABLE 3. Correlation between clinical variables and the renal ACE to ACE2 expression ratio ACE/ACE2
FIG. 1. Relationship between ACE and ACE2 mRNA levels in renal tissues. The mRNA levels were adjusted as relative values to GAPDH mRNA. To normalize the data distribution for statistical analysis, logarithmic transformation was performed for ACE and ACE2 gene expressions (after adjustment with GAPDH) (r ⫽ 0.396, P ⬍ 0.001).
Age (yr) UNaE (mEq/gCr) UPE (mg/gCr) Ccr (ml/min) PRA (ng/ml䡠h) PAC (pg/ml) s-ACE (IU/liter)
r
P
0.10 ⫺0.10 0.17 0.01 0.06 ⫺0.04 0.11
0.37 0.41 0.15 0.91 0.61 0.75 0.42
To normalize the data distribution for statistical analysis, square root transformation was performed for PRA, and logarithmic transformation was performed for UNaE, UPE, and ACE and ACE2 gene expressions (after adjustment with GAPDH).
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Wakahara et al. • ACE/ACE 2 Expression Ratio in Human Renal Tissue
TABLE 4. Clinicopathological variables and the renal ACE to ACE2 expression ratio Variables (n)
Gender Male (38) Female (40) HT ⫺ (58) ⫹ (20) Glomerular sclerosis scoreb I (36) II (29) III and IV (12) Tubulointerstitial scorec I (25) II (39) III (13)
ACE/ACE2 Mean ⫾
P
SD
0.76 ⫾ 0.22 0.67 ⫾ 0.21
0.07
0.69 ⫾ 0.20 0.80 ⫾ 0.25
0.049a
0.74 ⫾ 0.24 0.69 ⫾ 0.20 0.70 ⫾ 0.20
0.38 (vs. II) 0.96 (vs. III, IV) 0.54 (vs. I)
0.68 ⫾ 0.26 0.74 ⫾ 0.19 0.73 ⫾ 0.21
0.32 (vs. II) 0.88 (vs. III) 0.55 (vs. I)
P ⬍ 0.05. Glomerular injury was classified into four grades (I–IV) according to the method of Jackson and Johnston (25). c Tubulointerstitial injury was classified into three grades (I–III) according to the method of Bertani et al. (26). One of the samples was not adequate for glomerular assessment, and the other one was not adequate for tubulointerstitial assessment. To normalize the data distribution for statistical analysis, logarithmic transformation was performed for ACE and ACE2 gene expressions (after adjustment with GAPDH). a b
enzyme that cleaves Ang I and Ang II directly into Ang 1–9 and Ang 1–7, respectively, was identified (14, 15). This enzyme, a homolog of ACE, is designated as ACE2. ACE and ACE2 have been reported to be highly expressed in the endothelial cells of heart, kidney, and testis (14 –16). The ACE2 mainly degrades Ang II, which acts as a strong vasoconstrictor and is involved in cellular proliferation, to Ang 1–7, which is thought to act as a vasodilator and to be involved in apoptosis and growth arrest in contrast to ACE activity of generating Ang II and degrading Ang 1–7. Thus, ACE2 might be a potent counterregulator against ACE. Because local Ang II is thought to be a pivotal mediator in cardiovascular and renal injuries, ACE2 has emerged as a potential inhibitor of Ang II generation against the action of ACE. Recently some knowledge has been provided for functional roles of ACE2 in the cardiac and renal RAS (17–22). As an assessment of the human intrarenal RAS, there is only one report that neoexpression of ACE2 was found in several renal conditions based on nonquantitative histological evaluation so far (21) other than animal models (34, 35). The role and significance of ACE2 in the regulation of the RAS in human renal tissue remain to be well elucidated with quantitative evaluations. Moreover, clinical and pathological factors modulating the expression of ACE and ACE2 remain to be well elucidated. We already reported up-regulation of the ACE expression in diabetic nephropathy among major components of the RAS in our previous study (36). In this line, we first assessed the relationship between clinicopathological characteristics other than diabetes and the gene expression of ACE and ACE2 in human renal tissues. In the current study, we could not detect any significant relationship between clinicopathological characteristics, including HT, UPE, Ccr, and pathological grade of glomerular-interstitial changes, and each of the two gene expressions, being
assessed individually. Because we could detect a significant correlation between ACE and ACE2 gene expressions, we consider that this finding is very important for elucidating the mechanisms of the interplay between ACE and ACE2, and for establishing the role of ACE2 as a counterregulator against ACE. In particular, the significance of the results showing positive but not negative correlation would be controversial. Our data suggest the ACE2 gene expression is not actively involved in alteration of the local RAS status; rather, it can be speculated that ACE and ACE2 gene expressions may be regulated in a balanced manner, or synergistically, which is mediated via the local Ang II concentration. The exact mechanism for this regulation is not clear so far, and future study including transcriptional analysis would clarify the issue. A notable point here is that not only ACE but also ACE2 may be responsible for the local Ang II generation, whereas a balanced expression of ACE2 exerts a counteraction against ACE activities. With reference to the above speculation, the role of ACE2 must be taken into consideration for the pathogenesis of renal tissue injury. This synergistic expression has prompted us to test the possibility that some clinicopathological characteristics have substantial effects on the ACE to ACE2 gene expression ratio. Indeed, our assessment of the relationship between clinicopathological variables and the ACE to ACE2 ratio of renal tissue expression revealed that the ACE to ACE2 ratio is significantly influenced by the HT status. Whereas parameters of renal function, e.g. UPE and Ccr, are not significantly related with the ACE to ACE2 ratio as a whole (Table 3), the HT status may reflect disease-induced (or disease specific) deterioration of renal function. That is, UPE and Ccr of subjects with HT are significantly different from those without HT (UPE: 3.16 ⫾ 0.69 vs. 2.34 ⫾ 0.67, P ⬍ 0.0001; Ccr: 63.1 ⫾ 41.5 vs. 108.2 ⫾ 50.9, P ⬍ 0.0001, respectively), in which a significant correlation is also observed between UPE and Ccr (r ⫽ ⫺0.396, P ⬍ 0.001). Finally, stepwise regression analysis has further revealed that only the HT status is an independent determinant of the ACE to ACE2 ratio among major clinicopathological variables tested. Which of the HT status and ACE to ACE2 ratio is the cause and the result must be an issue. It seems that the HT status should be the cause for the alteration of the ratios; however, this is difficult to address and additional assessments, i.e. repeated biopsies and/or prospective follow-up, would be needed for solve the issue. Anyway, these results suggest that HT as a cardiovascular risk factor might influence the renal ACE to ACE2 expression ratio and thereby play a pivotal role in renal conditions, presumably, affecting the tissue concentrations of Ang II and Ang 1–7. In summary, the gene expressions of ACE and ACE2 were assayed with a very small amount of human renal tissues by quantitative methods and assessed for the relationship with clinicopathological characteristics. A significant correlation was observed between the gene expressions of ACE and those of ACE2. The ACE to ACE2 ratio is significantly influenced by the HT status. These findings suggest that besides ACE, ACE2, a newly emerging component of the RAS, is likely to play an important role in maintaining a balanced status of local RAS synergistically with ACE by counterregulatory effects confounded by the presence of hyperten-
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sion. Further investigation focusing also on ACE2 inhibitor (37) and ACE2 genetic variants (38) would enable us to better understand the roles of these enzymes (ACE and ACE2) in cardiovascular and renal conditions.
15.
Acknowledgments
17.
We are grateful to Professor Hiroyuki Nakamura (Department of Environmental Medicine, Kochi University School of Medicine, Kochi, Japan) for his advice on the statistical analysis. We are also grateful to Mr. John S. Gelblum for a critical reading of the manuscript and Miss Youko Hayashida for secretarial assistance.
18.
Received September 19, 2006. Accepted February 1, 2007. Address all correspondence and requests for reprints to: Tadashi Konoshita, M.D., Ph.D., Third Department of Internal Medicine, Fukui University School of Medicine, 23-3, Shimoaizuki, Eiheiji, Fukui, 9101193, Japan. E-mail:
[email protected]. This work was supported by Grants-in-Aid 08770879, 09770843, and 14571020 from the Ministry of Education, Science, and Culture of Japan and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (05-21). Disclosure Statement: The authors have nothing to disclose.
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References
24.
1. Hackenthal E, Paul M, Ganten D, Taugner R 1990 Morphology, physiology, and molecular biology of renin secretion. Physiol Rev 70:1067–1116 2. Corvol P, Soubrier F, Jeunemaitre X 1997 Molecular genetics of the reninangiotensin-aldosterone system in human hypertension. Pathol Biol (Paris) 45:229 –239 3. Zisman LS, Keller RS, Weaver B, Lin Q, Speth R, Bristow MR, Canver CC 2003 Increased angiotensin-(1–7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ACE2. Circulation 108:1707–1712 4. Sigmund C 2001 Genetic manipulation of the renin-angiotensin system in the kidney. Acta Physiol Scand 173:67–73 5. Vio CP, Jeanneret VA 2003 Local induction of angiotensin-converting enzyme in the kidney as a mechanism of progressive renal diseases. Kidney Int Suppl 86:S57–S63 6. Mezzano S, Droguett A, Burgos ME, Ardiles LG, Flores CA, Aros CA, Caorsi I, Vio CP, Ruiz-Ortega M, Egido J 2003 Renin-angiotensin system activation and interstitial inflammation in human diabetic nephropathy. Kidney Int Suppl 86:S64 –S70 7. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F 1990 An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 86:1343–1346 8. Danser AH, Schunkert H 2000 Renin-angiotensin system gene polymorphisms: potential mechanisms for their association with cardiovascular diseases. Eur J Pharmacol 410:303–316 9. McNamara DM, Holubkov R, Janosko K, Palmer A, Wang JJ, MacGowan GA, Murali S, Rosenblum WD, London B, Feldman AM 2001 Pharmacogenetic interactions between -blocker therapy and the angiotensin-converting enzyme deletion polymorphism in patients with congestive heart failure. Circulation 103:1644 –1648 10. Ortiz MA, De Prado A, Donate T, Gallart L, Claramunt H, Hernandez M, Martinez J, Martinez E, Pou JM 2003 Angiotensin-converting enzyme polymorphism gene and evolution of nephropathy to end-stage renal disease. Nephrology 8:171–176 11. Sakka Y, Babazono T, Sato A, Ujihara N, Iwamoto Y 2004 ACE gene polymorphism, left ventricular geometry, and mortality in diabetic patients with end-stage renal disease. Diabetes Res Clin Pract 64:41– 49 12. Samuelsson O, Attman PO, Larsson R, Mulec H, Rymo L, Weiss L, Ricksten A 2000 Angiotensin I-converting enzyme gene polymorphism in non-diabetic renal disease. Nephrol Dial Transplant 15:481– 486 13. Konoshita T, Miyagi K, Onoe T, Katano K, Mutoh H, Nomura H, Koni I, Miyamori I, Mabuchi H 2001 Effect of ACE gene polymorphism on age at renal death in polycystic kidney disease in Japan. Am J Kidney Dis 37:113–118 14. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart RE, Acton S 2000
25.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
37.
38.
2457
A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res 87:E1–E9 Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ 2000 A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem 275:33238 –33243 Harmer D, Gilbert M, Borman R, Clark KL 2002 Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme. FEBS Lett 532:107–110 Donoghue M, Wakimoto H, Maguire CT, Acton S, Hales P, Stagliano N, Fairchild-Huntress V, Xu J, Lorenz JN, Kadambi V, Berul CI, Breitbart RE 2003 Heart block, ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins. J Mol Cell Cardiol 35:1043–1053 Boehm M, Nabel EG 2002 Angiotensin-converting enzyme 2—a new cardiac regulator. N Engl J Med 347:1795–1797 Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveirados-Santos AJ, da Costa J, Zhang L, Pei Y, Scholey J, Ferrario CM, Manoukian AS, Chappell MC, Backx PH, Yagil Y, Penninger J 2002 Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417:822– 828 Goulter AB, Goddard MJ, Allen JC, Clark KL 2004 ACE2 gene expression is up-regulated in the human failing heart. BMC Med 2:19 Lely AT, Hamming I, van Goor H, Navis GJ 2004 Renal ACE2 expression in human kidney disease. J Pathol 204:587–593 Oudit GY, Herzenberg AM, Kassiri Z, Wong D, Reich H, Khokha R, Crackower MA, Backx PH, Penninger JM, Scholey JW 2006 Loss of angiotensinconverting enzyme-2 leads to the late development of angiotensin II-dependent glomerulosclerosis. Am J Pathol 168:1808 –1820 Ferrario CM, Jessup J, Gallagher PE, Averill DB, Brosnihan KB, Ann Tallant E, Smith RD, Chappell MC 2005 Effects of renin-angiotensin system blockade on renal angiotensin-(1–7) forming enzymes and receptors. Kidney Int 68: 2189 –2196 Kasahara Y, Ashihara Y 1981 Colorimetry of angiotensin-I converting enzyme activity in serum. Clin Chem 27:1922–1925 Jackson B, Johnston CI 1988 The contribution of systemic hypertension to progression of chronic renal failure in the rat remnant kidney: effect of treatment with an angiotensin converting enzyme inhibitor or a calcium inhibitor. J Hypertens 6:495–501 Bertani T, Cutillo F, Zoja C, Broggini M, Remuzzi G 1986 Tubulo-interstitial lesions mediate renal damage in adriamycin glomerulopathy. Kidney Int 30: 488 – 496 Shibata H, Suzuki H, Murakami M, Sato A, Saruta T 1994 Angiotensin II type 1 receptor messenger RNA levels in human blood cells of patients with primary and secondary hypertension: reference to renin profile. J Hypertens 12:1275–1284 Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G, Corvol P 1988 Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci USA 85:9386 –9390 Hubert C, Houot AM, Corvol P, Soubrier F 1991 Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene. J Biol Chem 266:15377–15383 Tso JY, Sun XH, Kao TH, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485–2502 Ercolani L, Florence B, Denaro M, Alexander M 1988 Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J Biol Chem 263:15335–15341 Dostal DE, Baker KM 1999 The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function? Circ Res 85:643– 650 Dzau V 2001 Tissue angiotensin and pathobiology of vascular disease: a unifying hypothesis. Hypertension 37:1047–1052 Tikellis C, Johnston CI, Forbes JM, Burns WC, Burrell LM, Risvanis J, Cooper M 2003 Characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy. Hypertension 41:392–397 Minghao Y, Jan W, Parveen N, Mohammad RS, Michael SL, Daniel B 2004 Increased ACE2 and decreased ACE protein in renal tubules from diabetic mice. Hypertension 43:1120 –1125 Konoshita T, Wakahara S, Mizuno S, Motomura M, Aoyama C, Makino Y, Kawai Y, Kato N, Koni I, Miyamori I, Mabuchi H 2006 Tissue gene expression of renin-angiotensin system in human type 2 diabetic nephropathy. Diabetes Care 29:848 – 852 Dales NA, Gould AE, Brown JA, Calderwood EF, Guan B, Minor CA, Gavin JM, Hales P, Kaushik VK, Stewart M, Tummino PJ, Vickers CS, Ocain TD, Patane MA 2002 Substrate-based design of the first class of angiotensinconverting enzyme-related carboxypeptidase (ACE2) inhibitors. J Am Chem Soc 124:11852–11853 Benjafield A, Wang W, Morris B 2004 No association of angiotensin-converting enzyme 2 gene (ACE2) polymorphisms with essential hypertension. Am J Hypertens 17:624 – 628
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