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Published online before print April 10, 2008
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From the Department of Medicine-Nephrology,* Baylor College of Medicine, Houston, Texas; the Department of Medicine, The University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong; and the Department of Pathology, Methodist Hospital, Houston, Texas
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Abstract |
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The recent discovery of the angiotensin II (Ang II) breakdown enzyme angiotensin I -converting enzyme (ACE)2 and alternative Ang II-generating pathways such as chymase, in addition to ACE, has increased the complexity of our understanding of Ang II generation and degradation in hypertension.2,3 ACE2 is a breakdown enzyme responsible for the degradation of Ang II to Angiotensin 1–7 peptide. The later has vasodilatory properties and has its own unique receptor, the Mas receptor.3 Emerging evidence shows that ACE2 plays an important role in negatively regulating hypertension. In rat models of hypertension, renal ACE2 mRNA and protein are decreased, although this could not be confirmed in human hypertensive nephropathy in a prior study.4 Further, in rats treated with ACE inhibitors and angiotensin receptor blockers, an increase in local renal ACE2 activity is noted.5,6 We have shown earlier that ACE is up-regulated in human diabetic nephropathy accompanied with hypertension, a condition associated with high Ang II levels.7 Taken together, these findings suggest that there may be an alteration in the ACE/ACE2 balance in hypertension in a manner that favors increased Ang II generation (ie, up-regulation of ACE) and decreased Ang II degradation (ie, down-regulation of ACE2) during hypertension.
The kidney is an important organ in hypertension. Not only is it a target for end organ damage in hypertension, but several lines of evidence suggest that it may play an active role in the pathogenesis of hypertension. For example, in salt-sensitive hypertension, renal blood flow is decreased with high salt diets8 and altered-pressure natriuresis curves have been described in essential hypertension.9 Reduced nephron numbers are also associated with essential hypertension and it is proposed that this may be associated with enhanced Ang II generation.10,11 Transplantation of kidneys from genetically hypertensive to normotensive rats results in hypertension in renal graft recipients.12 Conversely, kidneys from normotensive donors lower blood pressure in young transplanted spontaneously hypertensive rats.13 It has long been recognized that the renin angiotensin system is highly activated within the kidney during hypertension. Furthermore, it is also noted that ACE2 is expressed in normal rat kidney and is reduced in the rat kidney with hypertension. However, it is not known what factors down-regulate renal ACE2 during hypertension.
This study explored the signaling mechanism by which ACE2 is regulated under hypertensive conditions in vivo and in vitro. This was performed in patients with hypertensive cardiopathy and nephropathy and in vitro in a human tubular cell line (HK-2) in response to Ang II.
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Materials and Methods |
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Fetal bovine serum (FBS), penicillin/streptomycin/amphotericin B, Dulbecco’s modified Eagle’s medium, Dulbecco’s modified Eagle’s medium/F-12K medium, and insulin-transferrin-selenium were obtained from Invitrogen (Carlsbad, CA). Ang II, losartan, and PD 123319 (angiotensin II [AT]2 receptor antagonist) were obtained from Sigma (St. Louis, MO). Antibodies to ACE, ACE2, phosphorylated extracellular regulated (ERK)1/2, and phosphorylated p38 mitogen-activated protein (MAP) kinases were obtained from R&D systems (Minneapolis, MN). Antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Chemicon (Temecula, CA). ERK1/2 kinase inhibitor PD98059 and the p38 MAP kinase inhibitor SB203580 were purchased from Calbiochem (La Jolla, CA).
Heart and Kidney Tissue Collection and Immunohistochemistry
Specimens of normal human and hypertensive kidneys and hearts were obtained from the Department of Pathology, Methodist Hospital, following the approved protocol by Institutional Review Board of Baylor College of Medicine. Among them, 12 patients had been diagnosed with hypertensive nephropathy and 8 with hypertensive cardiomyopathy. All hypertensive patients (seven males and five females; 37 to 80 years of age) with unequivocal hypertension (systolic 141 ± 3.8 mm Hg) were treated with either angiotensin-converting enzyme inhibitor or AT1 receptor blockers. All patients had hypertensive history for up to 15 years. Both kidney and heart tissues were obtained at autopsy. In addition, 12 histologically normal kidneys were obtained from paratumor nephrectomy tissues and 8 normal cardiac tissues from among autopsy samples without cardiovascular diseases. Four-micron sections of the formalin-fixed, paraffin-embedded, human kidney and heart tissues were stained with antibodies to human ACE, ACE2, phosphorylated ERK1/2, and phosphorylated P38 MAP kinases in serial sections using the microwave-based antigen retrieval technique and a modified peroxidase anti-peroxidase method as described previously.7 Quantitative analyses of ACE and ACE2 expression within the kidney were performed using a quantitative image analysis system (Metamorph, Sunnyvale, CA). Briefly, up to five random areas (x10 power) were chosen from each tissue section and examined. The examined area was outlined, the positive staining patterns were identified, and the percent positive area in the examined area was then measured. Since ACE and ACE2 are expressed by all cardiac cells in normal and hypertensive heart tissues, the intensity score under low power-fields (x10) was used: (0.5) very weak expression with trace positive staining in most cardiac cells; (1) weak expression as determined by a clear but weakly positive immunostaining; (2) moderate expression as identified by positive signals between week and strong scores; and (3) strong expression as demonstrated by a marked immunostaining in most cardiac cells. For quantitative analysis of phospho-ERK1/2 and phospho-38, nuclear positive cells for pERK1/2 and pP38 were identified and percent positive nuclei were counted as previously described.14 Data were expressed as the percentage of mean ± SEM All examinations were performed blindly on coded slides.
Cell Culture
A human kidney tubular epithelial cell line (HK-2) was obtained from ATCC (Manassas, VA) and maintained in DMEM/F-12 containing 10% FBS. For all experiments, the cells were grown to confluence on 6- or 12-well plates (Falcon, Franklin Lakes, NJ) and made quiescent by incubation in serum-free DMEM for 24 hours before stimulation with Ang II. All reagents used were certified to be endotoxin free. Cells were stimulated with Ang II at 1 µmol/L for 0, 6, 12, 24, and 48 hours, and at doses of 0, 0.1, 0.25, 0.5, 1, 2, and 4 µmol/L for 24 hours, to examine the time and dose response of ACE and ACE2 expression. All cell cultures were performed in the presence of 0.5 mmol/L EDTA to prevent degradation of Ang II in the cell culture medium.
To inhibit binding of Ang II to its type I and type II receptor, losartan (1 µmol/L) and PD 123319 (1 µmol/L) were used. To inhibit Ang II induced ERK1/2 MAP kinase or p38 MAP kinase activities, inhibitors to ERK1/2 (PD98059, 20 µmol/L) or p38 (SB203580, 10 µmol/L) MAP kinases, and dominant negative (DN) Adv-DN-ERK or Adv-DN-p38 adenovirus were used, respectively. Recombinant adenovirus construct containing bacterial β-galactosidase gene (Adv-β-gal) was used as negative control. The characterization and transfection of these dominant negative vectors, as well as a negative control, has been well described elsewhere.14,15 Briefly, HK-2 cells were incubated with the adenovirus at multiplicity of infection of 30 in DMEM for 1 hour, and then made quiescent for 24 hours before stimulation with Ang II. Each experiment was repeated at least thrice throughout the study.
Reverse Transcription and Real-Time PCR
Total RNA from cell culture was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA) and total RNA from paraffin-sections of human kidney and heart samples was extracted by High Pure RNA Paraffin Kit (Roche Applied Science, Mannheim, Germany). Template cDNA was prepared using reverse transcriptase. Real-time PCR was performed with Sybgreen (Bio-Rad, Hercules, CA) and the Opticon real-time PCR machine (MJ Research Inc., Waltham, MA). The specificity of real-time PCR was confirmed via routine agarose gel electrophoresis and Melting-curve analysis. Housekeeping gene GAPDH was used as an internal standard. The primers used in this study are as follows: ACE forward 5'-GCAAGGAGGCAGGCTATGAG-3' and reverse 5'-CGGGTAAAACTGGAGGATGG-3'16 ; ACE2 forward 5'-CATTGGAGCAAGTGTTGGATCTT-3' and reverse 5'-GAGCTAATGC ATGCCATTCTCA-3'; GAPDH forward 5'-CAATGACCCCTTCATTGACC-3' and reverse 5'-GTTCACACCCATGACG AACATG.
Western Blot Analysis
Cultured HK-2 cells were lysed and protein was extracted, denatured at 99°C for 5 minutes, and then transferred to a nitrocellulose membrane. Nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% BSA in Tris-buffered saline buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The membranes were then incubated overnight at 4°C with primary antibodies against ACE, ACE2, phosphorylated ERK1/2, phosphorylated p38 MAP kinases, and GAPDH. After being washed extensively, the membranes then were incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature in 1% BSA/TBST. The signals were visualized by an enhanced chemiluminescence system (Amersham, Piscataway, NJ).
Statistical Analyses
All data are expressed as the mean ± SEM. Statistical significance was determined with one-way analysis of variance. t-tests were used for multiple comparisons. Differences were considered statistically significant at value of P < 0.05. Statistical analysis was conducted using STATA V6 (College Station, TX) and Microsoft Excel.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We first examined the hypothesis whether ACE is up-regulated and ACE2 is down-regulated in human hearts in hypertension. Serial sections of normal and hypertensive human hearts were examined for ACE and ACE2 expression by immunohistochemistry and real-time PCR. As shown in Figure 1
, serial sections of normal and hypertensive cardiopathy showed that there was constitutive ACE2 (Figure 1A)
and ACE (Figure 1B)
expression in the normal human heart. The presence of hypertension did not appear to significantly alter the level of ACE2 expression (Figure 1D)
, but up-regulated cardiac ACE (Figure 1E)
. This was further supported by a semiquantitative analysis (Figure 1G)
. Consistent with the immunohistochemical findings, real-time PCR demonstrated a twofold increase in mRNA expression of ACE in the hypertensive heart (Figure 1H)
. This was associated with a significant decrease in ACE2 mRNA expression (Figure 1H)
. Antibody controls are shown in Figure 1, C and F
.
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Immunohistochemically, serial sections of normal and hypertensive human kidneys revealed that a high expression of ACE2 was noted in the normal human kidney (Figure 2A)
. This was particularly strong in proximal tubular epithelial cells, glomerular epithelial cells, to a lesser extent in distal tubular epithelial cells, and vascular smooth muscle cells (Figure 2A)
. However, expression of ACE within the normal kidney was moderate (Figure 2B)
. In hypertensive nephropathy, ACE2 was markedly decreased, particularly in the tubules with severe tubulointerstitial damage (Figure 2F)
. Importantly, serial sections of immunohistochemistry demonstrated that a marked decrease in ACE2 in the hypertensive nephropathy was tightly associated with a strong up-regulation of ACE (Figure 2G)
, particularly in the damaged tubulointerstitium (Figure 2F vs 2G)
. A semiquantitative analysis of ACE and ACE2 expression in both normal and hypertensive kidney were shown in Figure 3A
. Indeed, a significant alteration of the balance of ACE:ACE2 from the normal level (ratio 1:3) to hypertensive nephropathy (ratio 3:1) was evident (Figure 3A)
.
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. This was associated with a dramatic reduction of ACE2 mRNA, virtually undetectable by real-time PCR (ACE2mRNA/GAPDH mRNA Ratio: 0.3073 ± 0.093 in the normal kidney versus 0.000002 ± 0.000001 in hypertensive nephropathy, Figure 3B
). Ang II Is Able to Up-Regulate ACE and Down-Regulate ACE2 Expression in Vitro
Since up-regulation of ACE and down-regulation of ACE2 were apparent in hypertensive nephropathy, a normal human kidney tubular epithelial cell line (HK-2) was used for studying mechanisms of ACE and ACE2 expression in response to Ang II. Quiescent HK-2 cells were stimulated with Ang II for varying time periods and at varying doses and the mRNA and protein were analyzed for ACE and ACE2 expression. As shown in Figure 4
, Ang II (1 µmol/L) induced a significant up-regulation of ACE mRNA (Figure 4A)
and protein (Figure 4, C and D)
in a time-dependent manner, peaking at 24 hours. This was associated with a significant suppression of ACE2 mRNA (Figure 4B)
and protein expression (Figure 4, C and E)
. Similarly, as shown in Figure 5
, both real-time PCR (Figure 5, A and B)
and Western blot analyses (Figure 5C)
showed that Ang II significantly up-regulated ACE and down-regulated ACE2 in both mRNA and protein levels in a dose-dependent manner, with the maximal effect at a dose of 1 µmol/L.
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Compared to normal kidney (Figure 2A–E)
, immunohistochemical staining in serial sections showed that up-regulation of ACE and down-regulation of ACE2 in hypertensive nephropathy (Figures 2, F and G, and 3A)
was associated with an increase in phosphorylated ERK1/2 (phospho-ERK1/2; Figures 2H and 3C
) and phosphorylated p38 (phospho-p38; Figures 2I and 3C
; Figure 2J
is a negative control) MAP kinases as demonstrated by their nucleated positive pattern. We further tested the hypothesis whether activation of ERK1/2 and p38 Map kinases is responsible for dysregulation of ACE and ACE2 expression in response to Ang II. HK-2 cells were stimulated with Ang II under the presence or absence of losartan (1 µmol/L, a selective AT1 receptor antagonist), PD123319 (1 µmol/L, a selective AT2 receptor antagonist), and inhibitors to ERK1/2 (PD98059, 20 µmol/L) or p38 (SB203580, 10 µmol/L) MAP kinases. As shown in Figures 6 and 7
, addition of losartan, but not PD123319, completely blocked Ang II-induced up-regulation of ACE and prevented the reduction of ACE2 in response to Ang II in both mRNA (Figure 6)
and protein (Figure 7)
levels. Interestingly, blockade of ERK1/2 or p38 MAP kinases with PD98059 or SB203580 prevented Ang II-induced down-regulation of ACE2 in both mRNA and protein levels, but not the effects on Ang II-induced up-regulation of ACE. These observations were further confirmed by inclusion of dominant negative ERK1/2 or p38 adenovirus. As shown in Figures 8 and 9
, blockade of ERK1/2 or p38 MAP kinases with Adv-DN-ERK and Adv-DN-P38 was able to abolish Ang II-induced down-regulation of ACE2, but not Ang II-induced up-regulation of ACE.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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There were several novel observations in this study. Firstly, the down-regulation of ACE2 expression and up-regulation of ACE in hypertensive nephropathy observed by both real-time PCR and immunohistochemistry suggested that the balance between these two enzymes was altered in hypertension. In the normal kidney, immunohistochemistry demonstrated that there was a threefold increase in ACE2 compared to ACE. However, there was a threefold decrease in ACE2, which was associated with a twofold increase in ACE in the hypertensive nephropathy. Consistent with the immunohistochemical finding, real-time PCR also showed a loss of ACE2 was associated with a marked up-regulation of renal ACE in hypertensive nephropathy. These observations imply that the balance between ACE and ACE2 is critical in the pathogenesis of hypertension in terms of Ang II generation versus degradation within the hypertensive kidney, although other Ang II-generating pathways may be also involved. In the normal kidney, constitutive high levels of ACE2 with higher ratio of ACE2/ACE (3:1) may be associated with an increase in the Ang II breakdown system compared to Ang II generation, which may be important in maintaining the normal physiological and biological effects of Ang II. In contrast, increased ACE and decreased ACE2 with higher ratio of ACE/ACE2 (3:1) in the hypertensive kidney may favor Ang II generation, leading to hypertensive cardiovascular and renal damage. This is consistent with our previous observation that ACE is up-regulated in diabetic nephropathy, particularly in those with hypertension.7 Indeed blockade of Ang II by either ACE inhibitors or angiotensin receptor blockers does improve progression of hypertensive cardiovascular and kidney diseases.24-26 Moreover, an association of increased Ang II degradation products with inhibition of ACE activities also suggests the importance of the balance of ACE/ACE2 in hypertension. The present study provides direct evidence that up-regulation of ACE is associated with the down-regulation of ACE2 in human hypertensive kidney disease. This implies that the alteration of ACE/ACE2 ratio may promote the disease progression in hypertension. However, it should be pointed out that there is limitation regarding the findings from human autopsy tissues. This is because the enzyme degradation occurs after death and it is unknown the time lag between the death and tissue collection in these patients. In addition, since all of the patients were treated with either ACE inhibitors or AT1 receptor blockers, it is difficult to exclude the potential effects of anti-hypertensive treatment on expression of ACE and ACE2 within the diseased kidney and heart. These may account for the discrepancy between mRNA and protein expression of ACE2 in the hypertensive heart, although this discrepancy may also be caused by the difference between the detecting methodologies used as discussed above. Nevertheless, the alteration of ACE/ACE2 ratio could be a valuable index for progression of hypertensive disease.
More importantly, we showed that Ang II was able to up-regulate ACE and down-regulate ACE2 in renal tubular epithelial cells and this occurred via the AT1 receptor, because blockade of AT1 receptor, but not AT2 receptor, abolished Ang II-induced up-regulation of ACE and down-regulation of ACE2. This novel finding suggests that there is an Ang II autoregulatory loop within the kidney, which is illustrated in Figure 10
. Indeed, the current view is that ACE and ACE2 act to counter-regulate one another.27,28
Down-regulation of ACE2 by ANG II represents a novel positive feed-forward system as noted in the brain.29
Once released, Ang II may signal through the AT1 receptor to up-regulate the ACE-dependent Ang II generating pathway and down-regulate the ACE2-mediated Ang II degradation pathway, ultimately leading to the elevation of Ang II levels and hypertension. Thus, Ang II could potentially upset the normal balance between the two ACEs, leading to conditions favoring excess Ang II generation and reduced Ang II breakdown. This would likely lead to more deleterious effects on progression of renal and cardiovascular disease, which commonly coexist in the setting of advanced hypertension and nephropathy. The magnitude order of dose-dependent effect of Ang II on up-regulation of ACE but down-regulation of ACE2 further supports this notion and implicates the interplay between two ACEs in local Ang II generation in response to Ang II. This is consistent with recent findings that loss of ACE2 or inhibition of ACE2 promotes glomerular injury in association with up-regulation of ACE expression in type-1 diabetic mice.30,31
Nevertheless, breakdown of this autoregulating pathway may be one mechanism of beneficial effects on hypertensive complications with ACE inhibitors and AT1 receptor antagonists.
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Furthermore, the present study also found that although both induction of ACE and suppression of ACE2 expression in response to Ang II occur through the AT1 receptor, disparate intracellular signaling pathways may operate. Ang II-induced down-regulation of ACE2 was mediated via the ERK1/2 and p38 MAP kinase pathways. This was supported by the observation that reduced ACE2 expression in response to Ang II was abolished by blocking ERK1/2 and p38 MAP kinases with both specific pharmacological inhibitors and Adv-DN-ERK and Adv-DN-P38. In contrast, blockade of ERK/p38 MAP kinases by either specific inhibitors or Adv-DN-ERK/p38 produced no effect on ACE expression in response to Ang II, indicating that an alternative Ang II signaling pathway may be responsible for ACE expression. Although the signaling pathways of ACE are well established,35 signaling mechanisms of Ang II –induced ACE remain largely unclear and require further investigation.
In summary, we have demonstrated that Ang II, once released, can act to up-regulate ACE but down-regulate ACE2 via the AT1 receptor-mediated mechanism. Activation of the ERK1/2 and p38 MAP kinase pathway may represent a key mechanism by which Ang II down-regulates ACE2.
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Footnotes |
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Supported by grants from the Research Grant Council of Hong Kong (RGC ERCG, HKU 759206, 768207), a Research and Conference Grant of The University of Hong Kong (2006–2007), and grants from the NIH (RO1HL076661, RO1DK062828, and T32DK062706).
Accepted for publication February 6, 2008.
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