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Renin inhibitor VTP-27999 differs from aliskiren: focus on their intracellular accumulation and the (pro)renin receptor

Lu, Xifeng; Krop, Manne; Batenburg, Wendy W.; Musterd-Bhaggoe, Usha M.; Garrelds, Ingrid M.; Danser, A.H. Jan

doi: 10.1097/HJH.0000000000000167
ORIGINAL PAPERS: Renin-angiotensin system

Background: VTP-27999 is a renin inhibitor with an IC50 that is comparable to that of aliskiren, but with a higher bioavailability. Unexpectedly, VTP-27999, unlike aliskiren, did not unfold renin's precursor, prorenin, and increased the affinity of the antibodies applied in renin immunoassays.

Methods: Here we verified to what degree these differences affect intracellular renin inhibitor accumulation in renin-synthesizing human mast cells (HMC-1), and (pro)renin's signaling via the (pro)renin receptor ((P)RR) in rat vascular smooth muscle cells. We also addressed the consequences of (P)RR knockdown by small-interfering (si) RNA on (pro)renin release. Finally, making use of FRET(Bodipy-FL)-labeled aliskiren, we studied, by subcellular fractionation, the cellular distribution pattern of this renin inhibitor.

Results: VTP-27999 accumulated at higher levels in HMC-1 cells than aliskiren, allowing this inhibitor to block intracellular renin at approximately five-fold lower medium levels. Labeled aliskiren accumulated in mitochondria and lysosomes, and its distribution pattern was different from that of renin. Moreover, the intracellular accumulation of both inhibitors in nonrenin-synthesizing HEK293 cells was not different from that in HMC-1 cells, suggesting that it is renin synthesis-independent. VTP-27999, but not aliskiren, blocked renin's capacity to stimulate extracellular signal-regulated kinase 1/2 phosphorylation in vascular smooth muscle cells, whereas neither inhibitor interfered with prorenin-induced signaling. (P)RR knockdown greatly increased renin (and to a lesser degree, prorenin) release, without affecting the capacity of forskolin or cAMP to stimulate renin release.

Conclusion: VTP-27999 differs from aliskiren regarding its level of intracellular accumulation and its capacity to interfere with renin signaling via the (P)RR, and the (P)RR determines prorenin–renin conversion and constitutive (but not regulated) (pro)renin release.

Division of Pharmacology and Vascular Medicine, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

Correspondence to Professor Dr A.H.J. Danser, PhD, Division of Pharmacology and Vascular Medicine, Department of Internal Medicine, Room EE1418b, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Tel: +31 10 7043540; fax: +31 10 7044733; e-mail:

Abbreviations: (P)RR, (pro)renin receptor; EKA, enzyme-kinetic assay; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; HMCs, human mast cells; IMDM, Iscove's modified Dulbecco's medium; IRMA, immunoradiometric assay; M6P, mannose 6-phosphate; RSA, relative specific activity; si, small interfering; VSMCs, vascular smooth muscle cells

Received 21 August, 2013

Revised 23 January, 2014

Accepted 8 February, 2014

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