New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

Elased, Khalid M.; Cunha, Tatiana Sousa; Gurley, Susan B.; Coffman, Thomas M.; Morris, Mariana

doi:10.1161/01.hyp.0000215588.38536.30

Cited by 69 publications

(81 citation statements)

References 41 publications

(80 reference statements)

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“…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.…”

Section: Discussionsupporting

confidence: 92%

Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II

Shaltout¹,

Westwood²,

Averill³

et al. 2007

American Journal of Physiology-Renal Physiology

129

151

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Despite the evidence that angiotensin-converting enzyme (ACE)2 is a component of the reninangiotensin 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 Ϯ 17 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ) and ACE2 [ANG II to ANG-(1-7): 253 Ϯ 11 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ], but lower neprilysin activity [ANG II to ANG-(1-4): 119 Ϯ 24 fmol ⅐ mg Ϫ1 ⅐ min Ϫ1 ; P Ͻ 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).ANG-(1-7); ANG-(1-5); neprilysin; ACE; ANG-(1-9) 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.Although the mechanisms for the generation and secretion of angiotensins within the proximal tubule are yet to be defined, the ...

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Section: Discussionsupporting

confidence: 92%

Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II

Shaltout¹,

Westwood²,

Averill³

et al. 2007

American Journal of Physiology-Renal Physiology

129

151

View full text Add to dashboard Cite

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“…That similar hemodynamic patterns were observed in diabetic ACE2 KO mice and wild-type mice receiving a selective ACE2 inhibitor suggests that circulating ACE2 may have an important role in the induction and maintenance of diabetic hyperperfusion. Although previous studies have failed to detect ACE2 activity in human plasma samples (26), the increased sensitivity of our assay and the ninefold higher ACE2 activity in murine samples compared with human plasma may partly explain this discrepancy. Moreover, the angiotensinase activity observed in this study was consistent with that of systemic ACE2, as it was inhibited by the same inhibitors (MLN-4760, EDTA, and Ang II; data not shown), and this enzyme activity was absent in ACE2 KO mice.…”

Section: Discussionmentioning

confidence: 56%

ACE2 Deficiency Modifies Renoprotection Afforded by ACE Inhibition in Experimental Diabetes

et al. 2008

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OBJECTIVE-The degradation of angiotensin (Ang) II by ACE2, leading to the formation of Ang 1-7, is an important step in the renin-angiotensin system (RAS) and one that is significantly altered in the diabetic kidney. This study examines the role of ACE2 in early renal changes associated with diabetes and the influence of ACE2 deficiency on ACE inhibitor-mediated renoprotection.RESEARCH DESIGN AND METHODS-Diabetes was induced by streptozotocin in male c57bl6 mice and ACE2 knockout (KO) mice. After 5 weeks of study, animals were randomized to receive the ACE inhibitor perindopril (2 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ). Wild-type mice were further randomized to receive the selective ACE2 inhibitor MLN-4760 (10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) and followed for an additional 5 weeks. Markers of renal function and injury were then assessed.RESULTS-Induction of diabetes in wild-type mice was associated with a reduction in renal ACE2 expression and decreased Ang 1-7. In diabetic mice receiving MLN-4760 and in ACE2 KO mice, diabetes-associated albuminuria was enhanced, associated with an increase in blood pressure. However, renal hypertrophy and fibrogenesis were reduced in diabetic mice with ACE2 deficiency, and hyperfiltration was attenuated. Diabetic wild-type mice treated with an ACE inhibitor experienced a reduction in albuminuria and blood pressure. These responses were attenuated in both diabetic ACE2 KO mice and diabetic mice receiving MLN-4760. However, other renoprotective and antifibrotic actions of ACE inhibition in diabetes were preserved in ACE2-deficient mice.CONCLUSIONS-The expression of ACE2 is significantly modified by diabetes, which impacts both pathogenesis of kidney disease and responsiveness to RAS blockade. These data indicate that ACE2 is a complex and site-specific modulator of diabetic kidney disease.

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“…In contrast to ACE, circulating ACE2 is low to nondetectable in rodents and humans, and tissue sources of the enzyme are more likely to influence the local RAS (12,40). Diabetic renal injury is generally associated with a reduction in the activity and/or expression of renal tissue ACE2, which may contribute to a deleterious imbalance in the relative expression of ANG II to Ang-(1-7) (21,26,39,52,53,60,63).…”

mentioning

confidence: 99%

Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes

Yamaleyeva¹,

Gilliam-Davis

Almeida³

et al. 2012

American Journal of Physiology-Renal Physiology

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Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB, Lindsey SH, Chappell MC. Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes. Am J Physiol Renal Physiol 302: F1374 -F1384, 2012. First published February 29, 2012 doi:10.1152/ajprenal.00656.2011We examined the impact of early diabetes on the circulating and kidney renin-angiotensin system (RAS) in male and female mRen2.Lewis (mRen2) hypertensive rats. Diabetes (DB) was induced by streptozotocin (STZ; 65 mg/kg) at 11 wk of age for 4 wk without insulin replacement. Systolic blood pressures were not increased in DB males or females compared with controls (CON). Circulating angiotensinconverting enzyme 2 (ACE2) increased ninefold (P Ͻ 0.05) in DB females and threefold (P Ͻ 0.05) in DB males, but circulating ACE and ANG II were higher in the DB groups. Serum C-reactive protein was elevated in DB females but not DB males, and the vascular responses to acetylcholine and estradiol were attenuated in the DB females. Proteinuria, albuminuria, and angiotensinogen excretion increased to a similar extent in both DB females and males. Glomerular VEGF expression also increased to a similar extent in both DB groups. Renal inflammation (CD68 ϩ cells) increased only in DB females although males exhibited greater inflammation that was not different with DB. Cortical ACE2 did not change in DB females but was reduced (30%) in DB males. Renal neprilysin activity (Ͼ75%, P Ͻ 0.05) was markedly reduced in the DB females to that in the DB and CON males. ACE activity was significantly lower in both female (75%, P Ͻ 0.05) and male (50%; P Ͻ 0.05) DB groups, while cortical ANG II and Ang-(1-7) levels were unchanged. In conclusion, female mRen2 rats are not protected from vascular damage, renal inflammation, and kidney injury in early STZ-induced diabetes despite a marked increase in circulating ACE2 and significantly reduced ACE within the kidney.angiotensin; kidney; neprilysin; hypertension; proteinuria ANGIOTENSIN-CONVERTING ENZYME 2 (ACE2), a homolog of ACE, is recognized as an important enzymatic component of the renin-angiotensin system (RAS) that may regulate functional output of this hormonal system (5,7,8,14,28,30,51,50). Although ACE2 was originally characterized for its ability to hydrolyze ANG I to Ang-(1-9), subsequent studies revealed a very high catalytic activity to convert ANG II to Ang-(1-7) (41,44,45,56,58). Indeed, ACE2 inhibition or genetic knockout studies reveal a heightened sensitivity to ANG II-induced increases in blood pressure, as well as an exacerbation of end-organ damage in these experimental models (31,32,46,48,52,60). In contrast to ACE, circulating ACE2 is low to nondetectable in rodents and humans, and tissue sources of the enzyme are more likely to influence the local RAS (12, 40). Diabetic renal injury is generally associated with a reduction in the activity and/or expression of renal tissue ACE2, which may contribute to a deleterious imbalance in the relative expression of ANG I...

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New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

Cited by 69 publications

References 41 publications

Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II

Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II

ACE2 Deficiency Modifies Renoprotection Afforded by ACE Inhibition in Experimental Diabetes

Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes

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