Kazal Boron Biswas

Aliskiren binds to renin and prorenin bound to (pro)renin receptor in vitro

Human (pro)renin receptor ((P)RR) has been implicated in the augmentation of many biological and cellular processes through bindings to its ligands, renin and prorenin. In this study, we investigated the effects of aliskiren, a direct oral renin inhibitor, on the activities of free and (P)RR-bound forms of human mature renin. We also elucidated the effect of aliskiren on the ‘renin activity’ of the receptor-bound form of prorenin. Aliskiren had an IC50 of 0.72 nmol l−1 against renin. The compound competitively inhibited renin activity with an inhibitory constant (Ki) of 0.18 nmol l−1. Furthermore, the dissociation constants (KD) for aliskiren from renin and prorenin bound to (P)RR were determined using surface plasmon resonance in a BIAcore assay system (Uppsala, Sweden). These values were estimated to be 0.46±0.03 and 0.25±0.01 nmol l−1, respectively. The compound competitively inhibited the renin activities of (P)RR-bound forms of both renin and prorenin with a Ki of 0.14 and 0.15 nmol l−1, respectively. These results indicate that aliskiren could be a potent inhibitor of the free forms of mature renin and of the receptor-bound forms of renin and prorenin.

Qualitative and quantitative analyses of (pro)renin receptor in the medium of cultured human umbilical vein endothelial cells

A 30-kDa protein in the medium of cultured human umbilical vein endothelial cells (HUVECs) was identified as (pro)renin receptor, (P)RR, by western blot analysis using anti-human (P)RR antibodies. The protein bound recombinant human prorenin with a KD of 4.0 nmol l−1 and activated prorenin. These observations suggest the presence of soluble (P)RR, s(P)RR, in the medium of cultured HUVECs. For quantification of the s(P)RR in the medium, an enzyme-linked immunosorbent assay (ELISA) was established. The quantitative range of the ELISA was validated over a nominal range of 7.5–300 pmol l−1 in the wells of a microtiter plate. The assay system showed good linearity (r2=0.99) with interassay (5.8–9.7%) and intraassay (2.1–7.0%) precision. Using this method, the concentration of s(P)RR in the culture medium of HUVECs was measured to be 32 pmol l−1. Therefore, these results show qualitative and quantitative evidence that prorenin can be activated after binding to s(P)RR secreted from cultured HUVECs.

Acid-activated prorenin binds to (pro)renin receptor in vitro

Binding properties of acid-activated prorenin to (pro)renin receptor [(P)RR] was investigated in vitro to discuss possible roles of such reversibly acid-activated prorenin in the renin angiotensin (RA) system. Prorenin was acidified at pH 3.3, 4.5, 5.5, 6.5, and its activation level was measured at 1, 2, 4, 8, 12, and 25 h. Prorenin, activated non-proteolytically in time- and pH-dependent manners, was verified by Western blot analyses. Acidification of prorenin for 25 h at pH 3.3, 4.5, 5.5, and 6.5 showed 78%, 54%, 34%, and 20% activities, respectively when compared with the renin activity of trypsinized prorenin as 100%. Additionally, the binding properties of acidified prorenin to (P)RR were elucidated both at the equilibrium state and in the kinetic state using BIAcore. BIAcore assay showed that acidified prorenin at pH 3.3, 4.5, 5.5, and 6.5 had apparent K(D) of 1.57 × 10(4), 14.1, 8.29, and 8.04 nM, respectively while native prorenin at pH 7.4 had a K(D) of 7.8 nM. At equilibrium state, K(D) of native prorenin was 1.42 nM whereas apparent K(D) varied from 1.25 to 5.0 nM for the prorenin acidified at pH 4.5, 5.5, and 6.5. The K(m) values of free forms of acidified prorenin at different pH (0.33-0.5 μM) was almost similar to those of (P)RR-bound forms of acidified prorenin (0.5-0.77 μM). These in vitro data indicate that prorenin acidified in vivo possibly modulate RA system in receptor-dependent and/or -independent manners which could ultimately lead to the pathogenesis of diseases.

Aliskiren reduces the release of soluble (pro)renin receptor from human umbilical vein endothelial cells

(Pro)renin receptor [(P)RR] has been implicated in diverse biological processes through binding to its ligands, which include renin, prorenin, Wnt signaling molecules and subunits of vascular H+-ATPase. Recent studies have reported that (P)RR is implicated in pathophysiological conditions including retinopathy and pancreatic ductal adenocarcinoma, and the soluble form of this receptor [s(P)RR] is considered as a useful biomarker for diseases. The present study examined the effect of aliskiren, the first orally active direct renin inhibitor, on the protein levels of (P)RR using cultured human umbilical vein endothelial cells (HUVECs). The cells were treated with or without aliskiren (10 nM) at 37°C for different durations (0, 8, 16 and 24 h). Aliskiren-treated HUVECs exhibited reduced proliferation compared with those treated without the drug. Furthermore, aliskiren treatment decreased not only the level of exogenous prorenin that bound to the membranes of HUVECs, but also the renin activity derived from this binding activity. These results indicate that the quantity of full-length (P)RR was reduced by aliskiren treatment, and furthermore, that the level of s(P)RR released from HUVECs was decreased with the treatment. Recent study has reported that s(P)RR exerted antidiuretic function. The current study suggests that the levels of s(P)RR, as a potential antidiuretic molecule and prospective disease biomarker, may be decreased during anti-hypertensive treatments with aliskiren.

A peptide YY inhibits the human renin activity in a pH dependent manner.

Royal jelly (RJ) is known to possess several physiological and pharmacological properties. A dipeptide YY derived from RJ proteins is known to inhibit angiotensin converting enzyme (ACE) activity. Our previous study showed that the dipeptide YY inhibited the human renin activity at physiological pH. In this study, we investigated the pH dependency of the inhibitory effect of the dipeptide YY to the renin reaction with angiotensinogen. The renin activity was expressed at a wide pH range with two peaks around at 6.0 and 8.0. The dipeptide YY was found to inhibit the renin activity only at the acidic pH range lower than 8.0. The Ki was estimated 4.6 micromol/L at pH 6.0 when the Km of human renin was determined 0.07 micromol/L using sheep angiotensinogen as the substrate. The Km was 0.25 micromol/L at pH 8.5. A stereo structure of the complex of human renin with the dipeptide YY was modeled to discuss its non inhibitory effect on the renin activity at the basic pH. It possibly owes to a local sift of YY space from the center of renin cleft into the N-domain side of renin molecule at basic pH range higher than 8.0.

Prorenin binding to (pro)renin receptor and its inhibition by the decoy peptide (RIFLKRMPSI) in vitro

Journal of the ReninAngiotensinAldosterone System