Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II.
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 x mg(-1) x min(-1)) and ACE2 [ANG II to ANG-(1-7): 253 +/- 11 fmol x mg(-1) x min(-1)], but lower neprilysin activity [ANG II to ANG-(1-4): 119 +/- 24 fmol x mg(-1) x 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).
Shaltout
Hossam A
HA
Department of Obstetrics and Gynecology, Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1095, USA.
Westwood
Brian M
BM
Averill
David B
DB
Ferrario
Carlos M
CM
Figueroa
Jorge P
JP
Diz
Debra I
DI
Rose
James C
JC
Chappell
Mark C
MC
D000803:Angiotensin I / Q000097:blood / Q000378:metabolism / Q000652:urine; D000804:Angiotensin II / Q000097:blood / Q000378:metabolism* / Q000652:urine; D000085962:Angiotensin-Converting Enzyme 2; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000809:Angiotensins / Q000097:blood / Q000378:metabolism* / Q000652:urine; D000818:Animals; D015153:Blotting, Western; D005260:Female; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000378:metabolism*; D015260:Neprilysin / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D012756:Sheep
D000806:Angiotensin-Converting Enzyme Inhibitors; D000809:Angiotensins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; C506331:angiotensin I (1-9); D000804:Angiotensin II; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; C000705328:Ace2 protein, mouse; C000705329:Ace2 protein, rat; D000085962:Angiotensin-Converting Enzyme 2; D015260:Neprilysin; C118790:angiotensin I (1-7)