Grobe N, Di Fulvio M, Kashkari N, Chodavarapu H, Somineni HK, Singh R, Elased KM. Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells. Am J Physiol Cell Physiol 308: C767-C777, 2015. First published March 14, 2015 doi:10.1152/ajpcell.00247.2014.-The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS. COS7; renin angiotensin system; ACE2 shedding; ADAM17 THE RENIN ANGIOTENSIN SYSTEM (RAS) plays a crucial role in blood pressure regulation and electrolyte balance (7). The RAS cascade is a multienzymatic system that begins with the formation of angiotensin I (ANG I) from hepatic angiotensinogen via renin (32). Angiotensin converting enzyme (ACE) cleaves the inactive decapeptide ANG I at the COOH-terminal dipeptide (L-histidyl-L-leucine), generating the potent vasopressor octapeptide ANG II (12). ANG II and ACE are involved in the initiation and progression of several diabetic complications such as retinopathy, nephropathy, hypertension, and cardiovascular disease (35). Blockade of the ANG II type 1 receptor (AT 1 R) augments renal plasma flow and suppresses filtration fraction in type II diabetic patients suggesting that a RAS blockade improves intrarenal hemodynamics (14). Furthermore, clinical trials have shown that blockade of RAS with ACE inhibitors (ACEIs) and AT 1 R blockers (ARBs) decreases the incidence of diabetes mellitus (13).The recently discovered ACE2, a homologue of ACE, provides a new target site for therapeutic intervention t...