Published online before print April 12, 2004, doi: 10.1161/01.HYP.0000126192.27644.76
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(Hypertension. 2004;43:1120.)
© 2004 American Heart Association, Inc.
Scientific Contributions |
Increased ACE 2 and Decreased ACE Protein in Renal Tubules From Diabetic Mice
A Renoprotective Combination?
From the Division of Nephrology and Hypertension (M.Y., J.W., P.N., M.R.S., M.S.L., D.B.), Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, Ill, and VA Chicago Health Care System/Lakeside Division (M.S.L., D.B.), Chicago, Ill.
Correspondence to Dr Daniel Batlle, Division of Nephrology and Hypertension, The Feinberg School of Medicine, Northwestern University, 320 E. Superior, Chicago, IL 60611. E-mail d-batlle{at}northwestern.edu
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Abstract |
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Unlike the ubiquitous angiotensin-converting enzyme (ACE), the ACE-related carboxypeptidase 2 (ACE 2) is predominantly expressed in the heart, kidney, and testis. ACE 2 degrades angiotensin (Ang) II to Ang (1–7) and Ang I to Ang (1–9). We investigated the expression of ACE and ACE 2 in a rodent model of type 2 diabetes. ACE and ACE 2 were measured in kidney and heart from 8-week-old no diabetic control (db/m) mice and diabetic (db/db) mice, which at this young age have obesity and hyperglycemia without nephropathy. In renal cortical tissue, ACE mRNA was reduced (db/db 0.31±0.06 versus db/m 0.99±0.05; P<0.005), whereas ACE 2 mRNA was not (db/db 0.94±0.05 versus db/m 1.03±0.11, NS). ACE protein was markedly reduced in kidney cortex of db/db mice (db/db 0.24±0.13 versus db/m 1.02±0.12; P<0.005), and this was associated with a corresponding decrease in renal ACE activity (db/db 12.7±3.7 versus db/m 61.6±4.4 mIU/mg protein; P<0.001). ACE 2 protein, by contrast, was increased in kidneys from diabetic mice (db/db 1.39±0.14 versus db/m 0.53±0.04; P<0.005). An increase in ACE 2 protein and a decrease in ACE protein, respectively, were also seen by immunostaining of renal cortical tubules from the db/db mice. In heart tissue, there were no significant differences between db/db and db/m mice in either ACE mRNA and protein or ACE 2 mRNA and protein. We conclude that in young db/db mice, ACE 2 protein in renal cortical tubules is increased, whereas ACE protein is decreased. We propose that the pattern of low ACE protein coupled with increased ACE 2 protein expression may be renoprotective in early stages of diabetes.
Key Words: angiotensin-converting enzyme • renin-angiotensin system • diabetic nephropathy • obesity • mice • ACE-related carboxypeptidase 2
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Introduction |
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Alterations within the renin-angiotensin system (RAS) are considered to be pivotal for the development of diabetic complications, particularly diabetic renal disease and hypertension.1–4 The angiotensin-converting enzyme (ACE), a key element of RAS, is primarily a membrane-bound protein residing on the surface of epithelial and endothelial cells.5 Through its 2 catalytic domains, ACE cleaves the inactive precursor angiotensin (Ang) I to Ang II, which induces vasoconstriction, aldosterone release, and acts as growth modulator.5–7 Most tissue beds, including the kidney, express a local RAS that acts independently of the circulating system.7–9 There is also a growing body of evidence that implicates the more recently characterized peptides Ang (1–7) and Ang (3–8) as additional bioactive components of the RAS.10–12
Recently, a homologue of ACE, an ACE-related carboxypeptidase (ACE 2), has been identified in humans and rodents.13–15 It contains only a single enzymatic site that is capable of catalyzing Ang I to Ang (1–9). It also degrades Ang II to the vasodilator Ang (1–7) and this may counterbalance the Ang II-forming activity of ACE.15,16 In contrast to ACE, ACE 2 activity is not inhibited by ACE inhibitors.13
Previous studies using the streptozocin (STZ) model of diabetes revealed decreased renal expression of ACE.17–19 A recent study using this rat diabetic model showed a reduction in ACE 2 as well.18 These previous studies involved diabetic rats with advanced renal lesions.17–19 The aim of the present study was to characterize the expression of ACE and ACE 2 in kidney from diabetic mice (db/db) before the development of nephropathy. The db/db mouse is a genetic model of type 2 diabetes caused by an inactivating mutation of the leptin receptor gene that results in a shorter intracellular domain of the receptor and a failure to transduce signals.20,21 As a result of this mutation, hyperglycemia develops in association with insulin resistance and obesity at 
4 to 7 weeks after birth.22 The db/db mouse eventually has some, but not all, features of human diabetic nephropathy such as renal hypertrophy, glomerular enlargement, and albuminuria.22,23 Renal histology evaluation, moreover, shows expansion of extracellular matrix as well as augmented laminin chain content.24,25 These lesions, however, are not present early on, but develop in older animals (20 weeks of age).22 We elected to study young db/db mice with short duration of diabetes to avoid potential confounding variables such as renal involvement and hypertension, which could directly or indirectly affect the expression and activity of ACE and ACE 2. We further examined ACE and ACE 2 expression in heart tissue as a way to determine if the altered expression of ACE and ACE 2 is organ-specific in diabetes.
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Methods |
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Animal Model and Biochemical Measurements
Diabetic mice (db/db) were used as a model of type 2 diabetes and their lean littermates (db/m) served as nondiabetic controls (Jackson Lab, Bar Harbor, Me). The db/db mouse is one of the best characterized and most extensively studied rodent models of type 2 diabetes.20 Heterozygous db/m littermates are lean and are spared from the induction of type 2 diabetes and its secondary complications.22 As such, it is an ideal genetic control for the db/db mouse. We used only young (age 8 weeks) female db/db mice to study an early phase of diabetes (3 to 4 weeks of onset) without renal complications.22 The Institutional Animal Care and Use Committee approved all procedures.
RNA Isolation and Reverse-Transcription Polymerase Chain Reaction
Total RNA was extracted from mice kidney cortices, hearts, and lungs with TRIZOL Reagent (Invitrogen). cDNAs were synthesized from 1.0 µg of total RNA by using Access RT-PCR system (Promega) as per manufacturer’s instructions and GenAmp PCR System 9700 (Applied Biosystems). The primers used for ACE were: 5'-TAACTCGAGTGCCGAGGTG-3' (sense) and 5'-CCAGCA-GGTGGCAGTCTT-3' (antisense), corresponding to nucleotide positions 200 to 218 and 522 to 539, respectively (ACC #BC040404). ACE 2 primers were: 5'-CTT CAGCACTCTCAGCAGACA-3' (sense) and 5'-CAACTTCCTCCTCACATAGGC-3' (antisense), corresponding to nucleotide positions 489 to 509 and 899 to 919, respectively (ACC #BC026801). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for each polymerase chain reaction (PCR). GAPDH primers were: 5'-CCAGTATGACTCCACTCACGGCA -3' (sense) and 5'-ATACT-TGGCAGGTTTCTCCAGGCG-3' (ACC #NM008084). The bands corresponding to PCR products were measured by densitometry.
Membrane Protein Preparation and Western Blot Analysis
Membrane proteins from kidney cortices and hearts were isolated and subjected to Western blot analysis as previously described.26,27 For detection of ACE, nitrocellulose membranes were incubated with mouse monoclonal antibody (Chemicon). ACE 2 protein in kidney tissue was measured using an affinity purified rabbit anti-ACE 2 antibody.15 For heart tissue, we used a commercial ACE 2 antibody (Santa Cruz). Signals on Western blots were quantified by densitometry and corrected for ß-actin,
ACE Activity Assay
Isolated kidney cortices, hearts, and lungs were homogenized in an assay buffer consisting of (in mmol/L) 50 HEPES, pH 7.4, 150 NaCl, 0.5% Triton X-100, 0.025 ZnCl2, and 1.0 PMSF, and then clarified by centrifugation at 10 000g for 15 minutes. ACE activity against a synthetic substrate (p-hydroxybenzoyl-glycyl-L-hisidyl-L-leucine) was determined using a colorimetric method (Fujirebio Inc). For the assay, tissue samples were standardized to 1 µg protein/µL. Optical density was read at 505 nm with a spectrophotometer. Results were calculated as mIU per mg of protein. All data are reported as mean±SE.
Immunohistochemistry
Kidneys were cut and fixed in 10% buffered formalin and embedded in paraffin. Sections (4 µm) were deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was performed with a pressure cooker at 120°C in target retrieval solution (DAKO). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Slides were then incubated with the same antibodies as described (anti-ACE or anti-ACE 215), and with secondary antibody conjugated with peroxidase-labeled polymer (DAKO). After incubation with DAB+ chromogen, slides were counterstained with hematoxylin. Sections were dehydrated, covered with Permount (Fisher Scientific) and a coverslip, and viewed with a Zeiss microscope.
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Results |
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Animal Characteristics
The basic animal characteristics are shown in the Table. As expected, db/db mice were much heavier than their lean db/m littermates and had markedly elevated serum glucose levels. Serum cholesterol and triglycerides were also markedly increased. Kidney weight was increased in db/db mice whereas the kidney-to-body weight ratio was reduced in db/db mice, likely reflecting their larger size.
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Reverse-Transcription PCR
Tissue levels of ACE mRNA were determined by semiquantitative RT-PCR after normalization against GAPDH. A single transcript of 339 bp was amplified for ACE and 624 bp for GAPDH (Figure 1). ACE/GAPDH ratio in renal cortex from db/db mice (n=5) was markedly lower than that observed in db/m controls (n=6) (db/db 0.31±0.06 versus db/m 0.99±0.05; P<0.005; Figure 1A). In contrast, ACE/GAPDH mRNA ratio in heart tissue was not different between db/db mice and control db/m mice (db/db 0.78±0.03 n=5 versus db/m 0.80±0.03, n=5, NS; Figure 1B). In lung tissue, there were also no significant differences between diabetic and control mice (db/db 0.97±0.11 n=5 versus db/m 0.91±0.05 n=6, NS).
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Tissue levels of ACE 2 mRNA were only determined in kidney cortex and heart, because lung tissue does not appear to express significant amounts of ACE 2.13 A single band at 430 bp was amplified by RT-PCR using ACE 2-specific primers (Figure 2). ACE 2/GADPH ratio in the kidney was not significantly different between diabetic db/db and db/m control mice (db/db mice 0.94±0.05 n=5 versus db/m 1.03±0.11 n=5, NS; Figure 2A). Likewise, in the heart, ACE 2/GADPH ratio was similar in db/db and db/m mice (db/db 0.70±0.06 versus db/m 0.81±0.07; NS; Figure 2B).
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ACE Activity
ACE activity was determined in renal cortex, heart, and lung tissue. ACE activity in the renal cortex was markedly decreased in diabetic mice compared with controls (db/db 12.7±3.7 versus db/m 61.6±4.4 mIU/mg protein, P<0.001; Figure 3A). In heart tissue, by contrast, ACE activity was similar in db/db and db/m mice (heart: db/db 1.81±0.26 versus db/m 2.05±0.21 mIU/mg protein, NS; Figure 3B). In lung tissue, ACE activity was the highest but not significantly different between db/db (269.9±32.9 mIU/mg protein) and db/m mice (229.5±19.6 mIU/mg protein). Thus, the reduction in ACE activity in diabetic mice appears to be organ specific for the kidney.
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Western Blotting
In kidney cortex and heart tissue, a single band of protein was seen at 170 kDa for ACE and at 89 kDa for ACE 2 when membranes were probed with the respective antibodies (Figures 4 and 5
). This is consistent with the molecular weights of ACE and ACE 2, respectively, as reported by others.28,29
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ACE protein expression was markedly reduced in kidney cortex of db/db mice as compared with that from db/m controls (db/db 0.24±0.13 n=5 versus db/m 1.02±0.12 n=5, P<0.005; Figure 4A). ACE 2 protein, by contrast, was higher in kidney cortex of db/db mice than in controls (db/db 1.39±0.14 n=5 versus db/m 0.53±0.04 n=5 P<0.005; Figure 4B). In heart tissue, there were no significant differences between db/db and db/m mice in either ACE (db/db 0.56±0.07 n=5 versus db/m 0.49±0.06 n=5) (Figure 5A) or ACE 2 protein abundance (db/db 0.72±0.07 n=5 versus db/m 0.79±0.11 n=5) (Figure 5B).
Immunohistochemistry
Prominent ACE and ACE 2 staining was observed in the renal cortex but not in the medulla. Strong staining for both ACE and ACE 2 was seen along the lumens of renal cortical tubules (Figure 6). There was a marked reduction in ACE staining in diabetic mice (Figure 6B) as compared with control mice (Figure 6A). By contrast, ACE 2 staining in cortical tubules of db/db mice was much more intense than in cortical tubules from the db/m controls (Figure 6D and 6C, respectively). These findings are in full concordance with the reduction in ACE protein and the increase in ACE 2 protein as determined by Western blotting.
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Discussion |
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In the present study, we investigated ACE and ACE 2 expression in the kidney and heart from young db/db mice, an animal model of type 2 diabetes. We found that the relative abundance of ACE 2 protein determined by Western blotting or by immunostaining was increased in kidney cortex from the db/db mice. ACE protein expression, by contrast, was profoundly decreased in renal tubules from the db/db mice as compared with nondiabetic controls. The reduction of tissue ACE protein expression and the augmentation in ACE 2 protein expression in db/db mice were limited to the kidney cortical tubules because no differences were observed between db/db and db/m mice in heart tissue.
The recently identified ACE homolog, ACE 2, differs from ACE in that it preferentially removes carboxy-terminal hydrophobic or basic amino acids.13,14 ACE 2 is highly expressed in kidney and heart.13 ACE 2 appears to be important in cardiac function as its deficiency results in severe impairment of cardiac contractility.15 To our knowledge, there is no evidence of cardiac dysfunction in the db/db mice in early stages of diabetes. In our study, ACE 2 mRNA and protein levels in the heart of diabetic mice were similar to control mice, which would be consistent with the lack of cardiac involvement at this stage.
In the db/db mice, the decrease in renal cortex ACE protein expression and increase in ACE 2 protein expression detected by Western blotting were fully concordant with the changes observed by immunostaining of renal cortical tubules. Prominent staining of ACE and ACE 2 was observed along the apical surface of cortical tubules in diabetic and control mice (Figure 6). The reduction of ACE in renal cortical tubules was unlikely to be caused by the loss of intact renal proximal tubules, which are the sites of the highest ACE concentration in the kidney,30 or ACE-bearing epithelial cells, because kidney histology in diabetic mice did not demonstrate any apparent structural abnormalities (data not shown). The finding of normal histology is consistent with previous studies in young mice with this model of diabetes.22 Intrarenal reduction of both metalloproteinases was recently described 24 weeks after diabetes induction using streptozocin.18 Differences in the results of that study and our findings regarding renal ACE 2 may be caused by differences in the diabetic model (type 1 versus type 2) or, more likely, in our opinion, disease duration and therefore absence of nephropathy at an early age (8 weeks).
Our finding of increased ACE 2 protein expression in renal cortical tubules from the young db/db mice with early diabetes does not exclude the possibility of an ACE 2 reduction later during the course of the disease as nephropathy develops. In fact, we speculate that with time, decreased ACE 2 expression with increased ACE may foster kidney damage in diabetes. ACE 2 cleaves Ang I to form Ang (1–9) and Ang II to form Ang (1–7).13–15,31 ACE 2 thus prevents Ang II accumulation while favoring Ang (1–7) formation. Ang (1–7) has vasodilatory, natriuretic, and antiproliferative actions.10–12 Its enhanced formation may have a beneficial effect and counterbalance the deleterious actions of Ang II in terms of kidney damage. Thus, the impact of a low ACE and a high ACE 2 protein levels on renal angiotensin peptides is apt to downregulate the renal RAS, which is felt to be overactive in the diabetic kidney. We therefore propose that the combination of low ACE and high ACE 2 protein expression in the kidney may provide renoprotection by attenuating Ang II accumulation and increasing Ang (1–7) formation. Interestingly, our finding that in young db/db mice the decrease in ACE activity was associated with an increase in ACE 2 protein expression resembles the pattern seen after administration of a renoprotective drug, ramipril, to diabetic rats.18
Renal ACE expression in db/db mice was reduced at all levels examined (mRNA, protein, and enzymatic activity) and to approximately the same extent (70% to 80%), likely reflecting downregulation at the transcriptional level. Renal ACE 2 mRNA, by contrast, was not different from controls, whereas ACE 2 protein was clearly increased. The mechanism by which ACE 2 protein is increased in the presence of normal mRNA levels was not investigated, although enhanced posttranscriptional processing could explain these observations. It would be interesting to examine if the marked suppression of ACE triggers a posttranscriptional increase in ACE 2 translation or decreased degradation in the kidney.
At age 8 weeks, the diabetic animals in our study already had severe obesity and hyperglycemia. It is unlikely that obesity in the db/db mice is responsible for the finding of suppressed renal ACE expression, because the opposite effect (ie, a kidney-specific increase in ACE activity) has been reported in obesity-prone mice when fed a high-fat diet.32 Thus one could speculate that hyperglycemia downregulates renal ACE transcription in db/db mice. This would be in keeping with the streptozotocin-treated rat model of diabetes in which kidney ACE mRNA was also decreased.17–19 Against this possibility, however, is our finding of a lack of changes in cardiac ACE mRNA and ACE protein despite exposure to a hyperglycemic environment similar to that prevailing in the kidney.
Perspectives
We have shown that in the young db/db mice, there is a profound reduction in the renal expression of ACE, whereas ACE 2 protein expression is increased at this stage of diabetes. The significance of a reduction in renal ACE coupled with an increase in ACE 2 protein expression needs to be clarified, but it seems reasonable to propose that such combination would attenuate angiotensin II formation and could exert a protective effect against the development of nephropathy at early stages of diabetes. Low renal ACE activity would be expected to limit Ang II formation, whereas an increase in ACE 2 should further prevent AngII accumulation by favoring conversion of Ang I to Ang (1–9) and Ang II to Ang (1–7). Because Ang II overactivity is thought to play a pivotal role in the progression of diabetic nephropathy, we suggest that decreased renal ACE activity coupled with increased renal ACE 2 expression may be protective for the kidneys in the early phases of diabetes by limiting the renal accumulation of Ang II and possibly by favoring Ang (1–7) formation as well.
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Acknowledgments |
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We thank Dr Mark C. Chappell and Dr Carlos M. Ferrario at the Wake Forest University School of Medicine for generously providing the ACE 2 antibody used for immunostaining and Western blots of kidney tissue. During the performance of these studies, D.B. was funded by grants from National Institutes of Health (RO-1 DK534460A2) and a Merit Review from the Department of Veterans Affairs.
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Footnotes |
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These studies were presented, in part, at the ASN Annual Meeting 2003 and at the High Blood Pressure Council Annual Meeting 2003.
Received December 19, 2003; first decision February 6, 2004; accepted March 10, 2004.
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 D. W. Wong, G. Y. Oudit, H. Reich, Z. Kassiri, J. Zhou, Q. C. Liu, P. H. Backx, J. M. Penninger, A. M. Herzenberg, and J. W. Scholey Loss of Angiotensin-Converting Enzyme-2 (Ace2) Accelerates Diabetic Kidney Injury Am. J. Pathol., August 1, 2007; 171(2): 438 - 451. [Abstract] [Full Text] [PDF]
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A. Gupta, G. J. Rhodes, D. T. Berg, B. Gerlitz, B. A. Molitoris, and B. W. Grinnell Activated protein C ameliorates LPS-induced acute kidney injury and downregulates renal INOS and angiotensin 2 Am J Physiol Renal Physiol, July 1, 2007; 293(1): F245 - F254. [Abstract] [Full Text] [PDF]
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 S. Wakahara, T. Konoshita, S. Mizuno, M. Motomura, C. Aoyama, Y. Makino, N. Kato, I. Koni, and I. Miyamori Synergistic Expression of Angiotensin-Converting Enzyme (ACE) and ACE2 in Human Renal Tissue and Confounding Effects of Hypertension on the ACE to ACE2 Ratio Endocrinology, May 1, 2007; 148(5): 2453 - 2457. [Abstract] [Full Text] [PDF]
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 F. A Ronchi, M.-C. Irigoyen, and D. E Casarini Association of somatic and N-domain angiotensin-converting enzymes from Wistar rat tissue with renal dysfunction in diabetes mellitus Journal of Renin-Angiotensin-Aldosterone System, March 1, 2007; 8(1): 34 - 41. [Abstract] [PDF]
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S. Keidar, A. Strizevsky, A. Raz, and A. Gamliel-Lazarovich ACE2 activity is increased in monocyte-derived macrophages from prehypertensive subjects Nephrol. Dial. Transplant., February 1, 2007; 22(2): 597 - 601. [Abstract] [Full Text] [PDF]
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 S. Keidar, M. Kaplan, and A. Gamliel-Lazarovich ACE2 of the heart: From angiotensin I to angiotensin (1-7) Cardiovasc Res, February 1, 2007; 73(3): 463 - 469. [Abstract] [Full Text] [PDF]
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 I. F. Benter, M. H. M. Yousif, C. Cojocel, M. Al-Maghrebi, and D. I. Diz Angiotensin-(1-7) prevents diabetes-induced cardiovascular dysfunction Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H666 - H672. [Abstract] [Full Text] [PDF]
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 M. Ye, J. Wysocki, J. William, M. J. Soler, I. Cokic, and D. Batlle Glomerular Localization and Expression of Angiotensin-Converting Enzyme 2 and Angiotensin-Converting Enzyme: Implications for Albuminuria in Diabetes J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3067 - 3075. [Abstract] [Full Text] [PDF]
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S. Giunti, D. Barit, and M. E. Cooper Mechanisms of Diabetic Nephropathy: Role of Hypertension Hypertension, October 1, 2006; 48(4): 519 - 526. [Full Text] [PDF]
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 J. Wysocki, M. Ye, M. J. Soler, S. B. Gurley, H. D. Xiao, K. E. Bernstein, T. M. Coffman, S. Chen, and D. Batlle ACE and ACE2 Activity in Diabetic Mice. Diabetes, July 1, 2006; 55(7): 2132 - 2139. [Abstract] [Full Text] [PDF]
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G. Y. Oudit, A. M. Herzenberg, Z. Kassiri, D. Wong, H. Reich, R. Khokha, M. A. Crackower, P. H. Backx, J. M. Penninger, and J. W. Scholey Loss of Angiotensin-Converting Enzyme-2 Leads to the Late Development of Angiotensin II-Dependent Glomerulosclerosis Am. J. Pathol., June 1, 2006; 168(6): 1808 - 1820. [Abstract] [Full Text] [PDF]
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U. Danilczyk and J. M. Penninger Angiotensin-Converting Enzyme II in the Heart and the Kidney Circ. Res., March 3, 2006; 98(4): 463 - 471. [Abstract] [Full Text] [PDF]
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 F. J. Warner, R. A. Lew, A. I. Smith, D. W. Lambert, N. M. Hooper, and A. J. Turner Angiotensin-converting Enzyme 2 (ACE2), But Not ACE, Is Preferentially Localized to the Apical Surface of Polarized Kidney Cells J. Biol. Chem., November 25, 2005; 280(47): 39353 - 39362. [Abstract] [Full Text] [PDF]
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 I. Fleming, K. Kohlstedt, and R. Busse New fACEs to the Renin-Angiotensin System Physiology, April 1, 2005; 20(2): 91 - 95. [Abstract] [Full Text] [PDF]
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N. Li, J. Zimpelmann, K. Cheng, J. A. Wilkins, and K. D. Burns The role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules Am J Physiol Renal Physiol, February 1, 2005; 288(2): F353 - F362. [Abstract] [Full Text] [PDF]
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