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作者：Hossam A. Shaltout, Brian M. Westwood, David B. Averill, Carlos M. Ferrario, Jorge P. Figueroa, Debra I. Diz, James C. Rose, and Mark C. Chappell作者单位：Department of Obstetrics and Gynecology, Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina
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【摘要】
~) {$ U" C" \& G* w" _; Z! J7 e Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the renin-angiotensin system (RAS), the influence of ACE2 on angiotensin metabolism within the kidney is not well known, particularly in experimental models other than rats or mice. Therefore, we investigated the metabolism of the angiotensins in isolated proximal tubules, urine, and serum from sheep. Radiolabeled [ 125 I]ANG I was hydrolyzed primarily to ANG II and ANG-(1-7) by ACE and neprilysin, respectively, in sheep proximal tubules. The ACE2 product ANG-(1-9) from ANG I was not detected in the absence or presence of ACE and neprilysin inhibition. In contrast, the proximal tubules contained robust ACE2 activity that converted ANG II to ANG-(1-7). Immunoblots utilizing an NH 2 terminal-directed ACE2 antibody revealed a single 120-kDa band in proximal tubule membranes. ANG-(1-7) was not a stable product in the tubule preparation and was rapidly hydrolyzed to ANG-(1-5) and ANG-(1-4) by ACE and neprilysin, respectively. Comparison of activities in the proximal tubules with nonsaturating concentrations of substrate revealed equivalent activities for ACE (ANG I to ANG II: 248 ± 17 fmol·mg -1 ·min -1 ) and ACE2 [ANG II to ANG-(1-7): 253 ± 11 fmol·mg -1 ·min -1 ], but lower neprilysin activity [ANG II to ANG-(1-4): 119 ± 24 fmol·mg -1 ·min -1; P < 0.05 vs. ACE or ACE2]. Urinary metabolism of ANG I and ANG II was similar to the proximal tubules; soluble ACE2 activity was also detectable in sheep serum. In conclusion, sheep tissues contain abundant ACE2 activity that converts ANG II to ANG-(1-7) but does not participate in the processing of ANG I into ANG-(1-9). 6 H$ B/ u W4 k
【关键词】 ANG() ANG() neprilysin ACE ANG()
/ G/ `/ ?( ^1 ]/ G THE INFLUENCE OF the renin-angiotensin-aldosterone system (RAAS) on the kidney to regulate blood pressure, the development of hypertension, and the extent of renal injury is established. Approaches that block the RAAS comprising angiotensin-converting enzyme (ACE) inhibitors, AT 1 receptor, or aldosterone antagonists constitute powerful therapies to control hypertension and attenuate renal damage in experimental and clinical studies. The kidney is a key target for biologically active components derived from both the circulating and intrarenal RAAS systems ( 25 ). Within the kidney, the proximal tubular epithelium is the primary if not sole site for angiotensinogen synthesis and release into the tubular fluid and contributes to the enzymatic processing of angiotensin peptides ( 20, 26 ). The proximal tubule and other tubular elements of the nephron are also target sites for the actions of angiotensin peptides and aldosterone to influence sodium and water transport. Indeed, Davisson et al. ( 9 ) suggest that renal angiotensinogen contributes to the development of hypertension in a mouse model of human renin and proximal tubule angiotensinogen that does not exhibit a significant increase in circulating ANG II.
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Although the mechanisms for the generation and secretion of angiotensins within the proximal tubule are yet to be defined, the tubular epithelium contains a number of peptidases including ACE and neprilysin that process ANG I once the decapeptide is cleaved from angiotensinogen ( 18, 30, 31 ). ACE is the major ANG II-forming enzyme in the kidney, serum, and other tissues; however, a novel homolog of the enzyme termed ACE2 was recently identified by Donoghue et al. ( 10 ) and Tipnis et al. ( 34 ). Unlike ACE, ACE2 does not convert ANG I to ANG II and the enzyme is resistant to ACE inhibitors ( 34 ). ACE2 exhibits much greater catalytic activity for ANG II to form ANG-(1-7) rather than converting ANG I to ANG-(1-9), or acting on other ACE substrates including bradykinin, substance P, and enkephalin ( 35 ). Similar to ACE, the kidney contains significant ACE2 activity that is predominantly localized to the proximal tubules ( 17, 33 ). Moreover, Crackower et al. ( 8 ) reported that several hypertensive models exhibit reduced ACE2 expression in the kidney and that the ACE2 knockout mice exhibit significant glomerulosclerosis ( 27 ). Despite the growing evidence that ACE2 is a component of the RAAS, the influence of ACE2 on angiotensin metabolism within the kidney or other tissues is not well known particularly in experimental models other than rats ( 23 ) or mice ( 8, 27 ). In this regard, the current study undertook a comprehensive analysis of angiotensin metabolism with an emphasis on the role of ACE2 in isolated proximal tubules of the kidney from adult female sheep, as well as in the urine and serum. These studies utilized the sheep model as the kidney closely parallels the human kidney in development and function.
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+ \; j# R& G* m9 I; {' P7 KMATERIALS AND METHODS1 a/ ]9 G( ~- E& m) L2 k5 c% j `
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Animals. Studies were performed using tissues obtained from four adult female sheep that were weaned at 3 mo of age after spontaneous delivery and brought into the laboratory at 18 mo of age. Animals were maintained on a normal diet, with free access to tap water in our AALAC-approved facility with a 12:12-h light-dark cycle (lights on 7 AM to 7 PM). Sheep were synchronized in their estrous cycles with intravaginal progesterone-releasing implants (EAZI-BREED CIDR, Pharmacia-Upjohn, Kalamazoo, MI). Implants were removed after 12-14 days, and a bladder catheter was inserted under anesthesia. Seven days after implant removal, sheep were anesthetized with ketamine and halothane and blood was obtained from a venous catheter. The blood was centrifuged at 3,000 g for 20 min and the isolated serum was stored at -80°C. The urine was collected from the bladder via the catheter, concentrated 10-fold on a Millipore 5,000-Da cut-off filter with the metabolism assay buffer, and was immediately utilized for metabolism experiments or frozen at -80°C. Kidneys were removed immediately and renal cortex was dissected out on ice for isolation of proximal tubules. These procedures were approved by the Wake Forest University School of Medicine IACUC for animal care.6 u! f4 w' n* G$ o1 X/ Q
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Proximal tubule preparation. Proximal tubules were prepared from a modified method of Vinay et al. ( 38 ). Kidney outer cortex was minced into fine pieces and incubated with collagenase (1 mg/ml, CLS 1, Worthington) at 37°C in a water-jacketed flask for 60 min containing 100 ml of a Krebs-Henseliet buffer (KHB, 25 mM HEPES, 118 mM NaCl, 4.8 mM KCl, 0.96 mM KH 2 PO 4, 25 mM NaHCO 3, 0.12 mM MgSO 4, 2.55 mM CaCl 2, pH 7.4) with 100 µl/ml DNase. At the end of the digestion, ice-cold KHB containing 10% fetal calf serum (FCS) was added, the suspension was filtered through a nylon mesh (70 µm) and centrifuged at 500 g for 5 min at 4°C to pellet the tubules. The pellet was resuspended with 32 ml of ice-cold KHB/5% FCS and gently applied to an isotonic discontinuous Percoll gradient (Pharmacia) of 10-35% (vol/vol) with KHB/5% FCS and centrifuged at 15,000 g for 60 min at 4°C. The cell layer at a density of 1,063 (F4, proximal tubules) as determined by density beads was washed in 3 x KHB to remove the Percoll media. The tubules were immediately utilized for metabolism experiments or frozen at -80°C.$ Z3 z: L- [* j$ K# `. B. O
* v7 i4 K; U3 R# R6 [# G+ z) tDetermination of angiotensin metabolism. Metabolism assays were conducted at 37°C in 10 mM HEPES, 125 mM NaCl, 10 µM ZnCl 2, pH 7.4, with 50 µg protein of fresh proximal tubules, 50 µg of tubule membranes (centrifugation of homogenized frozen tubules at 28,000 g for 10 min at 4°C and suspension of the pellet in assay buffer), 50 µl of serum, or 7 µl of urine (50 µg protein) in a final volume of 1.0 ml with or without the indicated inhibitors, and 0.5 nM iodinated [ 125 I]ANG I or [ 125 I]ANG II. The reaction was stopped by addition of ice-cold 1.0% phosphoric acid, centrifuged at 16,000 g, and the supernatant was stored at -20°C. Samples were filtered before separation by reverse-phase high-performance liquid chromatography (HPLC) and the 125 I products were monitored by a Bioscan flow-through detector as described ( 12 ). Products were identified by comparison of retention times to [ 125 I]standard peptides. Peptides were iodinated by the chloramine T method and purified by HPLC (specific activity of 2,200 Ci/mmol) ( 3 ). The following inhibitors, based on previous studies to distinguish ACE2 activity using ANG II as a substrate ( 12, 13 ), comprised the inhibitor cocktail in the assay: amastatin (AM; 2 µM), bestatin (BS; 10 µM), chymostatin (CHYM; 10 µM), benzyl succinate (BSC; 10 µM), and para-chloro-mercuribenzoic acid (PCMB; 0.5 mM). We subsequently added lisinopril to block ACE activity, SCH39370 for neprilysin activity, Z-prolyl prolinal (ZPP) for prolyl oligopeptidase activity, or MLN4760 for ACE2 activity (all at 10 µM final concentration).4 R( I; b4 O- ?. Z) M2 g8 ^
( V$ Y' v. K2 }: M6 sWestern blot analysis. Immunoblots for ACE2 were determined with an NH 2 terminally directed rabbit polyclonal antibody (Hypertension and Vascular Disease Center no. A2405) ( 16 ). The membrane fraction of the proximal tubules or the concentrated urine was diluted in SDS/ -mercaptoethanol solution and applied to 10% SDS polyacrylamide gels (Bio-Rad, Hercules, CA) for 60 min at 120 V in Tris-glycine SDS. Proteins were transferred onto a PVDF membrane and blocked with 5% nonfat dry milk in 0.1% Tween 20 in TBS for 60 min at room temperature before incubation with the ACE2 antibody (1:5,000). Immunoblots were then resolved with Pierce Super Signal West Pico Chemiluminescent substrate (Chicago, IL) as described by the manufacturer and exposed to Amersham Hyperfilm ECL (Piscataway, NJ).6 h z! w$ f+ o% p7 D
0 q7 n% b4 ^" w! m9 l3 l! W5 p6 rMaterials. Angiotensin peptides were purchased from Bachem (Torrance, CA). Acetonitrile (Optima grade) was obtained from Fisher Scientific (Fair Lawn, NJ). Lisinopril, a converting enzyme inhibitor, was provided by Merck (West Point, PA). SCH 39370, a neprilysin inhibitor, was provided by Schering-Plough (Madison, NJ). The ACE2 inhibitor MLN4760 was provided by Millennium Pharmaceuticals (Baltimore, MD). ZPP was purchased from BioMol (Plymouth Meeting, PA). All other reagents were obtained from Sigma (St. Louis, MO).
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) G# C9 g% J! E- VStatistics. Differences in the generation of [ 125 I]peptides under various conditions were assessed by one-way ANOVA with Student-Newman-Keuls post hoc analysis. ANG II degradation assessed without or with the ACE2 inhibitor in the tubules or serum was fitted to a single exponential decay curve. All statistical analyses were performed with GraphPad Prism and Stat Mate programs (GraphPad Software, San Diego, CA). The criterion for statistical significance was set at P 1 O/ j3 @; {- Q; O- ^% |1 N6 B
+ l& j8 s! u; B% T. s6 x- ERESULTS
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Proximal tubule metabolism. We initially assessed the contribution of ACE2 activity to the metabolism of [ 125 I]ANG and [ 125 I]ANG II in freshly isolated proximal tubules from the sheep kidney under conditions that should only reveal ACE2 activity ( 12, 13 ). As shown in Fig. 1, A and B, incubation of [ 125 I]ANG II with the inhibitor cocktail lacking the ACE2 inhibitor MLN4760 (MLN) demonstrated substantial conversion of ANG II to ANG-(1-7) ( Fig. 1 A ), whereas incubation with [ 125 I]ANG I revealed no metabolism of it ( Fig. 1 B ) in the tubules. The addition of the ACE2 inhibitor abolished the formation of ANG-(1-7) from ANG II ( Fig. 1 C ), while having no effect on ANG I metabolism ( Fig. 1 D ). These studies demonstrate ACE2 activity in intact proximal tubules preferentially converts ANG II to ANG-(1-7).9 f- B a+ }6 m
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Fig. 1. Metabolism of [ 125 I]ANG I and [ 125 I]ANG II in sheep proximal tubules. [ 125 I]ANG I or [ 125 I]ANG II was incubated with 50 µg freshly isolated proximal tubules for 30 min at 37°C and the metabolites were separated by HPLC. A : [ 125 I]ANG II was hydrolyzed primarily to [ 125 I]ANG-(1-7) in the presence of the inhibitor cocktail that lacks only the ACE2 inhibitor. C : addition of the ACE2 inhibitor MLN4760 (10 µM) abolished the generation of [ 125 I]ANG-(1-7). [ 125 I]ANG I was not metabolized in the cocktail that lacks only the ACE2 inhibitor ( B ) or includes the ACE2 inhibitor ( D ). HPLC results are representative of 4 separate metabolism experiments.
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Given the prolonged time to isolate the proximal tubules and perform the metabolism studies, subsequent assays to compare ACE2 to other peptidase activities in the proximal tubules were determined in the membrane fraction prepared from tubules stored at -80°C. The chromatographs reveal that, similar to the intact tubules, the complete inhibitor cocktail essentially prevented the metabolism of ANG II and removal of the ACE2 inhibitor again revealed a prominent peak of ANG-(1-7) ( Fig. 2, A and B, respectively). In Fig. 2 C, absence of both the neprilysin (SCH) and ACE2 inhibitors (MLN) revealed peaks for ANG-(1-4) and ANG-(3-4), but a reduced peak of ANG-(1-7), demonstrating neprilysin activity in the tubules. Finally, removal of the ACE inhibitor lisinopril (LIS) results in a prominent peak of ANG-(1-5) consistent with the ability of ACE to metabolize ANG-(1-7) to the pentapeptide ( Fig. 2 D ) ( 5 ). The quantification of the peptidase activities for ANG II metabolism from four adult sheep tubule preparations is shown in Fig. 3 A. In the absence of any inhibitors ( -inhibitors ), the major metabolites formed were ANG-(3-4), ANG-(1-7), ANG-(1-4), and ANG-(1-5). The addition of the cocktail inhibitor without the ACE, ACE2, or neprilysin inhibitors ( -LIS/MLN/SCH ) did not reduce the overall rate of ANG II metabolism in the tubules, but markedly reduced ANG-(3-4) and ANG-(1-7) formation as well as increased ANG-(1-4) and ANG-(1-5) generation. The addition of SCH to inhibit neprilysin ( -LIS/MLN ) attenuated ANG-(1-4) and ANG-(3-4), but increased formation of ANG-(1-5) and ANG-(1-7). Subsequent addition of LIS ( -MLN ) to block ACE attenuated ANG-(1-5) formation and revealed ANG-(1-7) as the major product; however, the overall rate of ANG II metabolism was not changed. Both the formation of ANG-(1-7) and the extent of ANG II metabolism were markedly reduced by inclusion of the ACE2 inhibitor MLN ( inhibitors ) in the proximal tubules. Additional studies determined the effect of ACE2 inhibition alone on ANG II degradation in the tubules. As shown in Fig. 3 B, the ACE2 inhibition significantly reduced the rate of ANG II metabolism by over twofold at the 15- and 30-min time points, as well as increased the half-life ( t 1/2 ) from 7.0 to 19.9 min. The extent of ANG II metabolism, however, was not changed by 60 min in the presence of the MLN agent, suggesting that other enzymes such as neprilysin contribute to ANG II degradation in the tubules. Figure 3 C summarizes the ANG II processing in the proximal tubules membranes based on the inhibitor data. ANG II is converted to ANG-(1-7) by ACE2 and possibly a second carboxypeptidase; however, ANG-(1-7) is rapidly metabolized by ACE to ANG-(1-5). ANG II is also directly converted to ANG-(1-4) through neprilysin and the enzyme continues to hydrolyze both ANG-(1-4) and ANG-(1-5) to the di- and tripeptide fragments Val-Tyr and Val-Tyr-Ile, respectively.$ u& `' a1 I8 u) F7 v
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Fig. 2. Metabolism of [ 125 I]ANG II by sheep proximal tubule membranes. [ 125 I]ANG II was incubated with 50 µg of proximal tubules membranes for 30 min at 37°C and the metabolites were separated by HPLC. A : [ 125 I]ANG II was not metabolized in the complete inhibitor cocktail. B : removal of the ACE2 inhibitor (MLN) revealed a prominent peak of ANG-(1-7) in the tubule membranes. C : further removal of the neprilysin inhibitor (SCH) revealed peaks for ANG-(1-4) and ANG-(3-4), but a reduced peak of ANG-(1-7). D : removal of ACE inhibitor lisinopril (LIS) revealed a prominent peak of ANG-(1-5) and reduced ANG-(1-7). HPLC results are representative of 4 separate metabolism experiments.( X; u: i! C# l( V8 Q
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Fig. 3. Quantification of the metabolism of [ 125 I]ANG II in sheep tubule membranes. A : quantification of the peptidase activities for [ 125 I]ANG II metabolism from the sheep proximal tubule membranes expressed as the rate of metabolism products formed in fmol·mg -1 ·min -1. [ 125 I]ANG II was incubated with 50 µg of proximal tubules membranes for 30 min at 37°C in either no inhibitors ( -inhibitors ), the inhibitor cocktail lacking the ACE inhibitor lisinopril (LIS), ACE2 inhibitor MLN4760 (MLN) and neprilysin inhibitor SCH39370 (SCH; -LIS/MLN/SCH ), the cocktail lacking LIS and MLN ( -LIS/MLN ), the cocktail lacking MLN ( -MLN ), and in the presence of all inhibitors ( inhibitors ). Results are means ± SE; n = 4. B : influence of ACE2 inhibition alone on [ 125 I]ANG II integrity as assessed by HPLC in proximal tubules. Data are means ± SE; n = 3. * P * B, y3 J' s& b- Y% t0 |: Z& A; x
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In Fig. 4, we show the chromatographic profile for ANG I metabolism in the tubule membranes. The inhibitor cocktail ( Fig. 4 B ) effectively prevented the metabolism of ANG I in the membranes and removal of the ACE2 inhibitor ( Fig. 4 A ) did not reveal ACE2 formation of ANG-(1-9). Removal of the SCH inhibitor revealed peaks corresponding to ANG-(1-7), ANG-(1-4), and ANG-(3-4), but a reduced peak of ANG I ( Fig. 4 C ). Removal of LIS ( Fig. 4 D ) revealed peaks for ANG II and ANG-(1-5), but a smaller peak of ANG-(1-7). The quantification of the peptidase activities for ANG I in the tubule membranes is shown in Fig. 5 A. In the absence of inhibitors ( -inhibitors ), the major metabolites from ANG I were ANG-(3-4), ANG-(1-7), ANG-(1-4), ANG-(1-5), and ANG II. The addition of the inhibitor cocktail without the ACE, ACE2, and neprilysin inhibitors ( -LIS/MLN/SCH ) again reduced the peaks for ANG-(3-4) and ANG-(1-7) but increased peaks for ANG-(1-4) and ANG-(1-5); the overall rate of ANG I metabolism was not changed. The addition of the neprilysin inhibitor SCH ( -LIS/MLN ) almost abolished both ANG-(1-4) and ANG-(3-4) formation but increased the ANG II and ANG-(1-5) and slightly reduced the overall rate of ANG I metabolism. The addition of the ACE2 inhibitor MLN ( -LIS ) reduced both ANG-(1-7) and ANG-(1-5) formation, but now revealed ANG II formation as the major activity. The addition of the ACE inhibitor LIS ( inhibitors ) abolished ANG II production and markedly reduced the overall metabolism of ANG I. Figure 5 B summarizes the ANG I processing in the proximal tubules based on the inhibitor data. ANG I is converted to ANG II by ACE and to ANG-(1-7) by neprilysin and an unknown activity; however, ANG II is rapidly converted to ANG-(1-7) and ANG-(1-4) via ACE2 and neprilysin, respectively. ANG I was directly converted to ANG-(1-7) through neprilysin and the heptapeptide was further metabolized to ANG-(1-5) by ACE. In Fig. 5 C, the full-length immunoblot reveals a single 120-kDa band in the proximal tubule membranes ( lanes 4 - 6 ), comparable in size to the human ACE2 standard ( lane 1 ). The supernatant fraction of the proximal tubule preparation did not reveal ACE2 staining.) s9 B8 S6 m4 z, P7 z1 B' ~9 m
6 ^- \. G q* u% n3 f8 R# }+ }* bFig. 4. Metabolism of [ 125 I]ANG I in sheep proximal tubule membranes. [ 125 I]ANG I was incubated with 50 µg of proximal tubules membranes for 30 min at 37°C and the metabolites were separated by HPLC. A and B : [ 125 I]ANG I was not metabolized in the cocktail that lacks only the ACE2 inhibitor ( A ) or includes the ACE2 inhibitor MLN4760 ( B ). C : removal of the neprilysin inhibitor SCH39370 revealed peaks for ANG-(1-7), ANG-(1-4), and ANG-(3-4). D : removal of ACE inhibitor lisinopril revealed a prominent peak of ANG-(1-5) and reduced ANG-(1-7). HPLC results are representative of 4 separate metabolism experiments.8 H6 E3 b/ ^5 w5 a. v, A$ x
$ B2 {8 E6 R* }5 ?Fig. 5. Quantification of the metabolism of [ 125 I]ANG I by sheep tubule membranes. A : quantification of the peptidase activities for [ 125 I]ANG I metabolism from the sheep proximal tubule membranes measured as the rate of metabolism products formed in fmol·mg -1 ·min -1. [ 125 I]ANG I was incubated with 50 µg of proximal tubules membranes for 30 min at 37°C in either no inhibitors ( -inhibitors ), the inhibitor cocktail lacking ACE inhibitor lisinopril (LIS), ACE2 inhibitor MLN4760 (MLN) and neprilysin inhibitor SCH ( -LIS/MLN/SCH ), the cocktail lacking LIS and MLN ( -LIS/MLN ), the cocktail lacking LIS ( -LIS ), and in the presence of all inhibitors ( inhibitors ). Results are means ± SE, n = 4. B : scheme summarizes the possible metabolism pathways for [ 125 I]ANG I in sheep proximal tubule membranes. C : immunoblot analysis of ACE2 in sheep proximal tubule membranes reveals a single 120-kDa band. Lane 1, human ACE2 standard; lane 2, molecular weight markers; lane 3, supernatant fraction of proximal tubules; lanes 4, 5, and 6, membrane fraction of proximal tubules., g3 I" h: V. X
% {) G7 P* c1 `2 r* q; I5 dUrinary metabolism. We determined the peptidase activities for ANG I and ANG II in urine collected from the bladder of the same sheep used as the source of the proximal tubules. As shown in Fig. 6 A, the major peptidase products for ANG II metabolism ( -inhibitors ) in urine were ANG-(1-7), ANG-(1-4), and ANG-(1-5). The addition of inhibitors without blocking ACE, ACE2, and neprilysin ( -LIS/MLN/SCH ) reduced ANG-(1-7) formation as well as overall ANG II metabolism. The neprilysin inhibitor ( -LIS/MLN ) reduced the ANG-(1-4) formation while LIS tended ( -MLN ) to reduce ANG-(1-5) formation. The ACE2 inhibitor ( inhibitors ) reduced ANG-(1-7) formation as well as the overall rate of metabolism of ANG II. Figure 6 B shows that the major products for ANG I metabolism in urine with no inhibitors includes ANG II, ANG-(1-7), ANG-(1-4), ANG-(1-9), and ANG-(3-4). The inhibitors in the absence of ACE, ACE2, and neprilysin blockade ( -LIS/MLN/SCH ) essentially reduced the ANG-(3-4)-, ANG-(1-7)-, and ANG-(1-9)-forming activities. The addition of SCH ( -LIS/MLN ) reduced ANG-(1-4) and ANG-(1-7). Inclusion of LIS ( -MLN ) reduced but did not abolish ANG II-forming activity and attenuated the rate of ANG I metabolism in urine. The addition of the ACE2 inhibitor ( inhibitors ) did not reduce the ANG-(1-9)-forming activity nor alter the overall rate of ANG I hydrolysis ( Fig. 6 B ). Urinary peptidase activity for both ANG II and ANG I metabolism was markedly lower than that in proximal tubules as calculated per milligram of protein.6 Z1 p' }0 k# U
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Fig. 6. Quantification of the metabolism of [ 125 I]ANG II and [ 125 I]ANG I in sheep urine. The quantification of the peptidase activities for [ 125 I]ANG II and [ 125 I]ANG I ( A and B, respectively) metabolism from the sheep urine measured as the rate of metabolism products formation in fmol·mg -1 ·min -1. [ 125 I]ANG II or [ 125 I]ANG II ( A and B, respectively) was incubated with 7 µl of concentrated urine (10-fold, 50 µg protein) for 30 min at 37°C in either no inhibitors ( -inhibitors ), the inhibitor cocktail lacking ACE inhibitor lisinopril (LIS), ACE2 inhibitor MLN4760 (MLN) and neprilysin inhibitor SCH ( -LIS/MLN/SCH ), the cocktail lacking LIS and MLN ( -LIS/MLN ), the cocktail lacking MLN ( -MLN ), and in the presence of all inhibitors ( inhibitors ). Results are means ± SE; n = 4.! K: y) n* ^0 L& P9 v2 E
. y \, k! s! q" Y4 b2 |! RSerum metabolism. We also determined the peptidase activities for ANG I and ANG II in sheep serum. As shown in Fig. 7 A, in the inhibitor cocktail lacking the ACE inhibitor, ANG I was metabolized to ANG II and LIS abolished ANG II formation ( Fig. 7 B ). ANG II was metabolized to ANG-(1-7) in the cocktail inhibitor lacking the ACE2 inhibitor ( Fig. 7 C ). The addition of the ACE2 inhibitor MLN ( Fig. 7 D ) completely abolished the formation of ANG-(1-7). Figure 7 E reveals an approximately twofold difference in ACE activity compared with ACE2 in the sheep serum. Finally, we assessed the effect of ACE2 inhibition alone on ANG II metabolism in the serum fraction. Although the overall rate metabolism of ANG II metabolism was much less than that in the sheep tubules or urine, the ACE2 inhibitor increased ANG II t 1/2 from 155 to 339 min ( Fig. 7 F ). f/ z' P' d3 v6 r' G' ~
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Fig. 7. Metabolism of [ 125 I]ANG I and [ 125 I]ANG II in sheep serum. [ 125 I]ANG I or [ 125 I]ANG II was incubated with 50 µl serum for 30 min at 37°C and the metabolites were separated by HPLC. A : [ 125 I]ANG I was hydrolyzed primarily to [ 125 I]ANG II in the presence of the inhibitor cocktail that lacks only the ACE inhibitor. B : addition of the ACE inhibitor lisinopril abolished the generation of [ 125 I]ANG II. C : [ 125 I]ANG II was hydrolyzed primarily to [ 125 I]ANG-(1-7) in the presence of the inhibitor cocktail that lacks only the ACE2 inhibitor. D : addition of the ACE2 inhibitor MLN4760 abolished the generation of [ 125 I]ANG-(1-7). HPLC results are representative of 4 separate metabolism experiments. E : comparison of ACE and ACE2 activities in sheep serum. Data are means ± SE; n = 4. * P
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5 h( ~9 W: R( _6 B) P+ LDISCUSSION
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( B4 G5 z" d4 P# J7 G/ d2 B: cACE2 is a novel monocarboxypeptidase highly expressed in the proximal tubule epithelium of the kidney that may function within the RAAS to process ANG II to ANG-(1-7) or ANG I to ANG-(1-9) ( 14 ). Elucidation of the contribution of ACE2 to the renal processing of angiotensins is important given that the enzyme is reduced within the kidney of several hypertensive models and that both ANG II and ANG-(1-7) have actions on the proximal tubule ( 4 ). The present results reveal that ANG II is converted to ANG-(1-7) by ACE2 within the proximal tubules of the sheep kidney. Following the formation of ANG-(1-7), the heptapeptide is rapidly metabolized to ANG-(1-5) by ACE, similar to the pathway in the circulation ( 41 ). In regards to ANG I, ANG II was the primary product through ACE and was subsequently converted to ANG-(1-7) by ACE2. ANG-(1-7) was generated from ANG I by neprilysin; however, ACE2 did not utilize ANG I as a substrate to form ANG-(1-9). Comparable processing pathways to the proximal tubules for ANG II and ANG I were demonstrated in urine, suggesting similar activities for the membrane-bound and solubilized forms of both ACE2 and ACE ( 36 ). Finally, we demonstrate soluble ACE2 activity in serum that may contribute to the metabolism of ANG II in this compartment.
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The preferred conversion of ANG II to ANG-(1-7) by ACE2 in the sheep kidney is entirely consistent with kinetic studies on various peptide substrates by the human enzyme ( 36 ). Although our studies find that ACE2 inhibition augmented the levels of ANG II in the proximal tubules, other enzymes including neprilysin likely influence the peptide's metabolism. These data on the contribution of ACE2 are consistent with recent studies by Elased et al. ( 11 ) and our group that demonstrated ACE2-dependent conversion of ANG II to ANG-(1-7) in membrane fractions of mouse kidney and rat renal cortex ( 13 ), respectively. However, the characterization of ANG II metabolism in the proximal tubules of sheep markedly differs from that reported by Burns and colleagues ( 23 ) in the rat kidney. These investigators found no evidence that ACE2 or other peptidases metabolize ANG II in proximal tubule preparations or in perfused proximal tubule segments isolated from male Sprague-Dawley rats ( 23 ). In their study, ACE2 activity was clearly evident in the rat proximal tubules since the conversion of ANG I to ANG-(1-9) was sensitive to ACE2 inhibition ( 23 ). If rat ACE2 does exhibit different kinetic properties for ANG I and ANG II than sheep or human, then the role of ACE2 in the regulation of the RAAS may be quite different among species. Additionally, the results of the two metabolism studies have important implications for the role of ACE as well, particularly whether ACE is involved in the formation ( 10, 23 ) or degradation of ANG-(1-7) ( 5, 21, 41 ). Campbell et al. ( 1 ) found significant quantities of endogenous ANG-(1-9) in the rat kidney; however, neither ACE inhibition ( 1 ) nor combined ACE/AT 1 blockade (Pendergrass KD and Chappell MC, unpublished observations) attenuated renal ANG-(1-7) levels in the rat. Furthermore, ANG-(1-7) levels within the kidney were maintained in the tissue ACE knockout mice ( 24 ). These in vivo studies do not provide convincing evidence that ACE predominantly contributes to the formation of ANG-(1-7) from ANG-(1-9) in the kidney.
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The present study allows for the relative comparison of multiple peptidase activities contributing to the processing of angiotensin peptides in the proximal tubule, urine, and serum under nonsaturating substrate concentrations. Utilizing ANG I in the presence of other inhibitors, ACE-dependent production of ANG II was 248 fmol·mg -1 ·min -1 in tubular membranes. ACE2 activity, based on the conversion of ANG II to ANG-(1-7), was comparable at 253 fmol·mg -1 ·min -1. Although we did not measure endogenous levels of ANG II or ANG-(1-7) in the sheep renal tissue, the comparable activities of ACE2 and ACE suggest that ACE2 may contribute to the metabolism of ANG II and the subsequent formation of ANG-(1-7) in the proximal tubules. Importantly, we found ANG-(1-7)-forming activity from ANG II or ANG I in the proximal tubules and urine that was reduced by the inhibitor cocktail lacking the ACE2 inhibitor (see Figs. 5 and 6 ). The design of the current study precluded the identification of all enzymatic pathways in the tubules and focused on the processing preferences of known peptidases such as ACE2; however, the present data clearly suggest an activity distinct from ACE2 that may contribute to the direct formation of ANG-(1-7) within the sheep kidney. The addition of the inhibitor cocktail lacking ACE2, ACE, and neprilysin inhibitors reduced ANG-(1-7) formation compared with the "no inhibitors" condition ( Figs. 3 A, 5 A, and 6, A and B ). These data suggest that a portion of ANG-(1-7)-forming activity in the tubules and urine may be due to an enzyme other than ACE2. Studies to identify this enzyme are in progress, although our preliminary characterization (Shaltout HA and Chappell MC, unpublished observations) revealed no inhibition by ZPP, a selective inhibitor of prolyl oligopeptidase (PO, EC3.4.11.26) capable of converting both ANG I and ANG II to ANG-(1-7) ( 15, 19 ). In this regard, we previously described a cysteine peptidase-like activity distinct from PO in the membrane fraction of NG-108 neuronal cells that hydrolyzed ANG I or ANG II to ANG-(1-7) ( 7 ).
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The present studies revealed substantial ACE2 activity in the urine collected directly from the bladder. In the urine, soluble ACE2 constituted the major enzymatic pathway for the metabolism of ANG II to ANG-(1-7); however, we could not demonstrate ACE2-dependent formation of ANG-(1-9) from ANG I. The glycosylated form of ACE2 is 120 kDa and the enzyme is unlikely to be filtered into the tubular fluid. Thus, the significant levels of ACE2 in the urine most likely reflect release from the proximal epithelium. Lambert et al. ( 22 ) reported that the metallopeptidase ADAM 17 may function as a secretase to release ACE2 from the extracellular side of the cell membrane. Moreover, expression of ACE2 into Madin-Darby canine kidney cells exclusively trafficks to the apical side of these cells while ACE localizes to both the apical and basolateral aspects ( 28 ). The apical expression of ACE2 in the proximal epithelium would be consistent with the demonstration of ACE2 activity in sheep urine. We have previously documented that urine contains angiotensin peptides and ACE that are most likely derived from the kidney ( 39, 40 ). In this regard, Casarini and colleagues ( 2 ) suggest that the NH 2 -terminal form of ACE (80 kDa) may be a urinary marker for hypertension. Demonstration of ACE and ACE2 activities also suggests that these soluble enzymes may contribute to the tubular or urinary fluid levels of angiotensin, apart from formation along the apical membrane or within the renal epithelium. The proximal tubule contains ANG II receptors on both the apical and basolateral aspects of the epithelium. Thus, the localization of ACE2 may influence the expression of ANG II as well as its product ANG-(1-7), a peptide that generally opposes the actions of ANG II and recognizes a distinct receptor, the Mas protein ( 4, 29, 32 ). Finally, we demonstrate ACE2-dependent formation of ANG-(1-7) from ANG II in sheep serum. ACE2 activity was 50% lower than that of ACE, suggesting that the accumulation of ANG II may be preferred in the serum fraction of female sheep.
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) X1 S, @! {7 q) UIn conclusion, the current study documented a role of ACE2 and other peptidases localized to the sheep proximal tubules, urine, and serum to control levels of angiotensins. Both membrane-bound and soluble forms of ACE2 contributed to the conversion of ANG II to ANG-(1-7) in the proximal tubules, serum, and urine but not to the direct metabolism of ANG I. The extent that ACE2 plays a major role in the formation of ANG-(1-7) from ANG II in the sheep proximal tubules must be considered in lieu of other peptidases such as ACE and neprilysin that metabolize the heptapeptide in the proximal tubules. Indeed, this "peptidase triad" may determine the ultimate balance of ANG II and ANG-(1-7) following the formation of ANG I in the tubular environment, although it is quite likely that other enzymatic pathways exist in other renal and extrarenal compartments.) P2 ^2 [1 |: r q# E8 n
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GRANTS# a0 P9 V4 Z& j
6 l: `+ X, _1 P# c0 W0 O# SThis study was supported by National Heart, Lung, and Blood Institute Grants HL-56973, HL-51952, and HL-68728, National Institute of Child Health and Human Development Grant HD-047584 from the National Institutes of Health (Bethesda, MD), and unrestricted grants from Unifi (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, NC).1 Q* z, b* z7 D% g1 J
4 G0 U9 h& T& V% V0 l" e0 bACKNOWLEDGMENTS5 \! o4 J4 w- t* k' Y% C) w/ u
; l# g; l1 r8 C; E8 uWe gratefully acknowledge the gift of lisinopril from Merck, MLN 4760 from Millennium Pharmaceuticals, and SCH 39370 from Schering Plough.
2 v, m, X/ ?$ ]& ` 【参考文献】0 @, L f: q9 B8 O8 y0 {8 N
Campbell DJ, Lawrence AC, Towrie A, Kladis A, and Valentijn AJ. Differential regulation of angiotensin peptide levels in plasma and kidney of the rat. Hypertension 18: 763-773, 1991.0 X r: Z$ F j4 a, E/ S- G0 e% g" F
* L6 ~7 [( o7 L
( ~- u; k" E, R: ^1 O4 O
4 X3 @% v, D* [2 O `4 qCasarini DE, Boim MA, Stella RCR, and Schor N. Endopeptidases (kininases) are able to hydrolyze kinins in tubular fluid along the rat nephron. Am J Physiol Renal Physiol 277: F66-F74, 1999.8 I1 W, K4 r. y4 f. f
# w& d) r: T# ~
( Y2 R* q3 E6 @) W7 O$ H
' n' ~4 s9 A2 }+ A4 @/ |; ?% WChappell MC, Brosnihan KB, Diz DI, and Ferrario CM. Identification of angiotensin-(1-7) in rat brain: evidence for differential processing of angiotensin peptides. J Biol Chem 264: 16518-16523, 1989.
5 J) I/ J1 }# K j+ s
1 \6 \! j2 X1 Z, z$ q4 q9 |1 L) h9 |6 r
' ?2 H9 w, ]4 z# }) y4 e
Chappell MC, Modrall JG, Diz DI, and Ferrario CM. Novel aspects of the renal renin-angiotensin system: angiotensin-(1-7), ACE2 and blood pressure regulation. In: Kidney and Blood Pressure Regulation, edited by Suzuki H and Saruta T. Basel: Karger, 2004, p. 77-89.$ Z7 x1 M5 ]: s' X c/ K/ y" F
3 \/ M. D8 s9 i$ Z
% A$ p$ r( h) U1 J
+ J4 w9 S& k; W7 z1 wChappell MC, Pirro NT, Sykes A, and Ferrario CM. Metabolism of angiotensin-(1-7) by angiotensin converting enzyme. Hypertension 31: 362-367, 1998." m3 m c* z- R9 C: A4 P
5 z# N: A t$ b( u9 I
) k7 U- q5 t( t8 S8 [% j
& i6 S6 t% \* n0 C9 X( p4 Y" r
Chappell MC, Tallant EA, Brosnihan KB, and Ferrario CM. Processing of angiotensin peptides by NG108-15 neuroblastoma X glioma hybrid cell line. Peptides 11: 375-380, 1990.
% e8 z- g) p2 S% @, N0 U* b+ h$ k
! M; T3 [: `' z3 C- A5 q n* ?( l, Z/ U% W# K ?
# e/ J% E4 d+ FCrackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira-dos-Santo AJ, da Costa J, Zhang L, Pei Y, Scholey J, Bray MR, Ferrario CM, Backx PH, Manoukian AS, Chappell MC, Yagil Y, and Penninger JM. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417: 822-828, 2002.
' J& u& M j& y6 b# ]4 {3 g# y2 {6 Y% k
' n7 H3 j/ p2 g
& E; f1 ^% J/ V! }8 F4 g: T
Davisson RL, Ding Y, Stec DE, Catterall JF, and Sigmund CD. Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice. Physiol Genomics 1: 3-9, 1999.
; b' w1 c+ P7 y' `
?* @& A. }* V* k. m6 A7 ~
, O7 S1 v6 @& d x9 m, {
( F& ~9 o7 d& J3 X9 G& b& \Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robinson K, Jeyaseelan R, Breitbart RE, and Acton S. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 87: E1-E9, 2000.
* H. `- H" j6 }8 C, P8 ]% [( s8 V* f! l$ Q8 f
) Q7 w# u3 U$ D
5 _3 }" p0 n: ] cElased KM, Cunha TS, Gurley SB, Coffman TM, and Morris M. New mass spectrometric assay for angiotensin-converting enzyme 2 activity. Hypertension 47: 1010-1017, 2006.
- S# z2 a( L+ X# \, w, m
% {& _2 ]/ }/ l5 u" {( v3 b5 y* u2 M. X6 w. i+ o e) i
% |1 j1 `$ u; a L1 V( q( @Ferrario CM, Jessup JA, Chappell MC, Averill DB, Brosnihan KB, Tallant EA, Diz DI, and Gallagher PE. Effect of angiotensin converting enzyme inhibition and angiotensin II receptor blockers on cardiac angiotensin converting enzyme 2. Circulation 111: 2605-2610, 2005.) K8 ]: [8 m- f; m' @8 h! p2 W
7 c3 W! H& n5 Q1 a
- p& ]. z+ |3 a, s! I
# F) q8 X4 ^9 B9 f
Ferrario CM, Jessup JA, Gallagher PE, Averill DB, Brosnihan KB, Tallant EA, Smith RD, and Chappell MC. Effects of renin angiotensin system blockade on renal angiotensin-(1-7) forming enzymes and receptors. Kidney Int 68: 2189-2196, 2005.
5 R2 |; g# R$ @+ z6 J A6 H2 c, | h* r) B3 A% e
# l u) s1 o0 R9 S6 q" D1 A2 u, t9 @/ X3 [) H
Ferrario CM, Trask AJ, and Jessup JA. Advances in the biochemical and functional roles of angiotensin converting enzyme 2 and angiotensin-(1-7) in the regulation of cardiovascular function. Am J Physiol Heart Circ Physiol 289: H2281-H2290, 2005.
5 C6 V( ?' j5 V$ t1 k/ ]3 {& ^5 `. u' W( R9 @" ]$ c. N
A, }# E0 }( ]0 X7 {' b+ ^ B, ~# T3 f( s' a$ n
Friedman TC, Orlowski M, and Wilk S. Prolyl endopeptidase: inhibition in vivo by N-benzyloxycarbonyl-prolyl-prolinal. J Neurochem 42: 237-241, 1984. @4 k0 a5 S3 J- q
( f% s* q4 W g5 H( B W* @, V, m
; W4 p3 x( z3 y7 j# ^( q
8 u9 @. m+ L8 j% V# H- B
Gallagher PE, Chappell MC, Ferrario CM, and Tallant EA. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol 290: C420-C426, 2006.$ Z/ j/ u4 j7 Z$ E: {
8 F# K, v) L* [$ d& ]: d; h7 W. \
7 R" t2 Z$ d5 X6 Y vGembardt F, Sterner-Kock A, Imboden H, Spalteholz M, Reibitz F, Schultheiss HP, Siems WE, and Walther T. Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents. Peptides 26: 1270-1277, 2005.
! b( D {' m v3 J
# u& ?6 O. Y9 q% L4 ]
/ I- y3 m: \' F M! Y0 v' C$ g' I# e. {5 |
George SG and Kenny AJ. Studies on the enzymology of purified preparations of brush border from rabbit kidney. Biochem J 134: 43-57, 1973.) a7 Z4 D) n( G! @8 _4 [
: W5 H+ C1 m/ y. W) _/ ?. s/ j: M7 v4 o2 a5 _. N% {2 { U
, m& u: |" c% G
Greene LJ, Spadaro ACC, Martins AR, Jesus WDP, and Camargo ACM. Brain endo-oligopeptidase B: a postproline cleaving enzyme that inactivates angiotensin I and II. Hypertension 4: 178-184, 1982.9 b: v$ [2 O1 ~* I$ [: O. V
2 t2 @- `+ [! R3 x4 A; }
! A: p" m* \. m9 N l" h
+ w8 N, X2 }* x; S2 \4 U
Hjemdahl P and Wiklund IK. Quality of life on antihypertensive drug therapy: scientific end-point of marketing exercise? J Hypertens 10: 1437-1446, 1992.
' }, ?+ X/ x. N4 |- L0 I4 N8 {3 t6 j# K, r/ R i. H, J
- P1 Q& f2 j- L0 b
* {2 p. e$ ^5 A9 OIyer SN, Ferrario CM, and Chappell MC. Angiotensin-(1-7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system. Hypertension 31: 356-361, 1998.2 r( J& j7 L5 L% |0 p' b! T
. q$ z$ w' D# k0 i9 [
' D: F* F. e3 {) X& a7 e4 b3 z% x% l; I3 l* u/ K0 L4 H! h
Lambert DW, Yarski M, Warner FJ, Thornhill P, Parkin ET, Smith AI, Hooper NM, and Turner AJ. Tumor necrosis factor- convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-Co-V) receptor, angiotensin-converting enzyme-2 (ACE2). J Biol Chem 280: 30113-30119, 2005.
7 l3 J- P; D3 m; X3 ] h) V+ g% N( T) y
! A# g) A. t8 I! e: ?2 G! `) o4 J" D# e; c. p5 p2 m6 P: P
3 p1 ^: |6 B- y$ b) a" k. L ZLi N, Zimpelmann J, Cheng K, Wilkins JA, and Burns KD. The role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules. Am J Physiol Renal Physiol 288: F353-F362, 2005.' ^( E1 K9 F8 D) E5 c/ ?+ F, @
+ a) L$ F( X$ W5 V( N4 {
; @/ N$ e! G; G* a
E; K' F7 e- p& p. |# I
Modrall JG, Sadjadi J, Brosnihan KB, Gallagher PE, Ya CH, Kramer GL, Bernstein KE, and Chappell MC. Depletion of tissue ACE differentially influences the intrarenal and urinary expression of angiotensins. Hypertension 43: 4849-4853, 2003./ [, C7 l9 ?' |0 ` y3 [
0 x4 m! h1 j# p' @$ N1 y" u
B( I8 H' S- |; F& _3 L
/ y; A+ E/ q7 |; q+ j: v ANavar LG, Harrison-Bernard LM, Nishiyama A, and Kobori H. Regulation of intrarenal angiotensin II in hypertension. Hypertension 39: 316-322, 2002.
( O* d' S) p3 ~+ }2 `, O- J5 W
I% I1 n0 D8 }% Z' r. [6 ]) l9 v9 U$ {5 }& j& ~
1 A+ T; `' G) y `' K
Navar LG, Lewis L, Hymel A, Braam B, and Mitchell KD. Tubular fluid concentrations and kidney contents of angiotensins I and II in anesthetized rats. J Am Soc Nephrol 5: 1153-1158, 1994.1 s6 r, _' U2 H) d/ ^7 G
4 \) [& I6 g% Z- }; U' p, p+ p9 g8 }- U# o; f7 |3 n7 {" M4 T- n
L( H' V. N8 h1 w( _$ t! R* }: GOudit GY, Herzenberg AM, Kassiri Z, Wong D, Reich H, Khokha R, Crackower MA, Backx PH, Penninger JM, and Scholey JW. Loss of angiotensin-converting enzyme-2 leads to the late development of angiotensin II-dependent glomerulosclerosis. Am J Pathol 168: 1808-1820, 2006.
3 j S& `- L2 _; U7 }6 A6 ?7 t0 ?/ e. c
- }0 _. K, \5 u( @3 ?, Y: }6 g# S6 c5 W
Rice GI, Thomas DA, Grant PJ, Turner AJ, and Hooper NM. Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism. Biochem J 383: 45-51, 2004.5 x2 A; G, `( z2 [5 ~1 u4 C
' l- `3 a* U* I0 I# J
( a$ J- U' M3 c+ l' c W2 p* ?0 f0 Y& s
Santos RAS, Silva ACS, Maric C, Silva DMR, Machado RP, de Buhr I, Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M, Mendes EP, Lemos VS, Campagnole-Santos MJ, Schultheiss HP, Speth R, and Walther T. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci USA 100: 8258-8263, 2003.+ G% i9 h" w1 ?6 M$ j" _3 I" x
( O4 n/ q0 J, e' }0 X: @9 @2 e' g, E. |4 X5 s
1 T& z; [. \+ P4 iStephenson SL and Kenny AJ. The metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes. Biochem J 241: 237-247, 1987./ L% k& Q" v1 ]5 L& L- s
2 {/ Z; R/ z0 n1 H/ z, N3 r0 R
, X; Z% y3 M+ o) A) |2 V4 a3 ^1 A% H* i! f5 u- i% k- E+ [
Sudo J and Tanabe T. Distributions of postproline cleaving enzyme-, converting enzyme-, trypsin- and chymotrypsin-like activities in various nephron segments and in brush-border membrances isolated from rat kidney. Chem Pharm Bull (Tokyo) 33: 1694-1702, 1985." Y* x9 [2 Z' F, x4 M1 O: b
; C L( u. }* `
* i: r2 |9 A' X3 G* v9 Y# z p
4 w. ?; c1 o) z+ X \. |6 {! jTallant EA, Ferrario CM, and Gallagher PE. Angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor. Am J Physiol Heart Circ Physiol 289: H1560-H1566, 2005.
' @5 f1 y3 V% b0 ~5 X4 ^2 ^& r; W4 H7 c9 q% g' ]
' a% G* y' c9 G3 H2 s- x. `
- K' V6 n& i. k D: I
Tikellis C, Johnston CI, Forbes JM, Burns WC, Burrell LM, Risvanis J, and Cooper ME. Characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy. Hypertension 41: 392-397, 2003.
! t/ j. ]4 R7 Q, p- e0 O2 j( A/ \* b8 w) C! N& _
/ C' i1 O9 f9 d$ b' l3 p2 M3 G8 ]* S- E/ _0 ~! K
Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, and Turner AJ. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidse. J Biol Chem 275: 33238-33243, 2000.
0 ?, D) M0 f5 q" ^
. c7 y6 v4 E( y* r
- F" ]9 Z: E9 f) d9 X
6 `' y0 R; e, }4 xVickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A, and Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277: 14838-14843, 2002.
2 y1 @5 e- `5 w" g, z; X
+ Q& ~; U4 A2 q) m
, e: l+ y% G1 m) p6 A! N# k! }! g( w o6 Y- ]
Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A, and Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277: 14838-14843, 2002.
1 X/ F9 v Q8 p; s, e! b: p" U6 w7 c7 ~
* v9 ?( y- a4 F. {& k( l& h- [/ l5 h {, m
0 g! f* w: B5 |% b* yVinay P, Gougoux A, and Lemieux G. Isolation of a pure suspension of rat proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 241: F403-F411, 1981.) Q f2 E, l: j w- l
# H1 f$ |! U. q2 }, t6 E* a- o3 g L" d F, |
$ g7 B5 @% x c, j# g
Wong DL, Chappell MC, Ferrario CM, Ganten D, and Diz DI. Spironolactone prevents activation of the intrarenal renin-angiotensin system and increases survival in (mREN2) 27 transgenic rats with malignant hypertension. Hypertension 36: 724, 2000.1 C8 S ]) c0 H7 @* A
+ L" [) b7 [ h3 D, h. ?; a' ?
* U" }' Y O. W% Z! O( X9 Z6 |" O2 v* j v, D( Q
Yamada K, Iyer SN, Chappell MC, Brosnihan KB, Fukuhara M, and Ferrario CM. Differential response of angiotensin peptides in the urine of hypertensive animals. Regul Pept 80: 57-66, 1999.
/ K* M( `0 m7 Z6 Y
' s6 I# x$ [% D% i' K# Q! @* t. {* `. D1 O6 j
( O" s: i( m) u% u/ j6 B
Yamada K, Iyer SN, Chappell MC, Ganten D, and Ferrario CM. Converting enzyme determines the plasma clearance of angiotensin-(1-7). Hypertension 98: 496-502, 1998. |
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