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

Abstract

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 ...

Angiotensin‐(1‐7): Pharmacology and New Perspectives in Cardiovascular Treatments

“…Recent studies confirmed that NEP can metabolize Ang I directly into Ang-(1-7), and, furthermore, NEP can act upon Ang II to yield Ang-(1-4) and Ang-(3-4), but not Ang-(1-7) in sheep renal proximal tubular membranes (Shaltout et al 2007). …”

Renal ACE2 expression and activity is unaltered during established hypertension in adult SHRSP and TGR(mREN2)27

“…28,29 Besides, kinetic differences in AngI and AngII metabolism are shown to be exist between rat, human and sheep kidneys. 25,30,31 There are some limitations to our study. First, our data were obtained in adult animals with established hypertension and hence do not rule out a role for renal ACE2 in the early onset of hypertension.…”

Renin-angiotensin system in the kidney: What is new?

“…It is widely accepted that small peptides derived from Ang Ⅱ have local physiological effects, especially in the kidney (Figure 1). ACE2 is a transmembrane glycoprotein that shares a 42% of homology with ACE and contains a single active site domain more closely to the N domain of ACE [16,38] . Unlike ACE, ACE2 is a monocarboxypeptidase, generating Ang-(1-7) by the cleavage of a single Phe residue from Ang Ⅱ, and Ang-(1-9), removing the C-terminal Leu residue from Ang Ⅰ [16,38] .…”

“…ACE2 is a transmembrane glycoprotein that shares a 42% of homology with ACE and contains a single active site domain more closely to the N domain of ACE [16,38] . Unlike ACE, ACE2 is a monocarboxypeptidase, generating Ang-(1-7) by the cleavage of a single Phe residue from Ang Ⅱ, and Ang-(1-9), removing the C-terminal Leu residue from Ang Ⅰ [16,38] . Within the renal brush-border vesicles of the rat, Ang-(1-7) is preferentially hydrolyzed by aminopeptidases and neprylisin (NEP) after aminopeptidase blockade, generating Ang-(1-4) [39] .…”

“…New RAS members and hypertension treatment renal NEP Ang I is hydrolyzed primarily to Ang-(1-7) and Ang-(1-4) [40,41] . In sheep proximal tubules, urine and serum, Ang Ⅱ is converted to Ang-(1-7) by both membrane-bound and soluble forms of ACE2 [38] . The physiological importance of Ang-(1-7) has become increasingly evident, especially after Santos et al [14] found a G protein-coupled receptor for Ang-(1-7), the Mas receptor, using a selective Ang-(1-7) antagonist.…”