5 research outputs found
High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin
Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II
(man-6-P/IgF-II) receptors are involved in the activation of recombinant
human prorenin by cardiomyocytes. To investigate the kinetics of this
process, the nature of activation, the existence of other prorenin
receptors, and binding of native prorenin, neonatal rat cardiomyocytes
were incubated with recombinant, renal, or amniotic fluid prorenin with or
without man-6-P. Intact and activated prorenin were measured in cell
lysates with prosegment- and renin-specific antibodies, respectively. The
dissociation constant (K(d)) and maximum number of binding sites (B(max))
for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and
3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin
internalization was greater than 10 times B(max). Levels of internalized
intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating
proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and
man-6-P-containing fractions revealed that only the latter was bound.
Cells also bound and activated renal but not amniotic fluid prorenin. We
concluded that cardiomyocytes display high-affinity binding of renal but
not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding
precedes internalization and proteolytic activation to renin thereby
supporting the concept of cardiac angiotensin formation by renal prorenin
Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors
ACE inhibitors improve endothelial dysfunction, possibly by blocking
endothelial angiotensin production. Prorenin, through its binding and
activation by endothelial mannose 6-phosphate (M6P) receptors, may
contribute to this production. Here, we investigated this possibility as
well as prorenin activation kinetics, the nature of the
prorenin-activating enzyme, and M6P receptor-independent prorenin binding.
Human umbilical vein endothelial cells (HUVECs) were incubated with
wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala,
thereby preventing cleavage by known proteases), M6P-free prorenin, and
nonglycosylated prorenin, with or without M6P, protease inhibitors, or
angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1
nmol/L, maximum number of binding sites [B(max)] 1010+/-50
receptors/cell). At 37 degrees C, because of M6P receptor recycling, the
amount of prorenin internalized via M6P receptors was >25 times B(max).
Inside the cells, wild-type and K/A-2 prorenin were proteolytically
activated to renin. Renin was subsequently degraded. Protease inhibitors
interfered with the latter but not with prorenin activation, thereby
indicating that the activating enzyme is different from any of the known
prorenin-activating enzymes. Incubation with angiotensinogen did not lead
to endothelial angiotensin generation, inasmuch as HUVECs were unable to
internalize angiotensinogen. Most likely, therefore, in the absence of
angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin
internalization by endothelial cells represents prorenin clearance
Plasma renin and prorenin and renin gene variation in patients with insulin-dependent diabetes mellitus and nephropathy
BACKGROUND: The most striking abnormality in the renin angiotensin system
in diabetic nephropathy (DN) is increased plasma prorenin. Renin is
thought to be low or normal in DN. In spite of altered (pro)renin
regulation the renin gene has not been studied for contribution to the
development of DN. METHODS: We studied plasma renin, prorenin, and four
polymorphic markers of the renin gene in 199 patients with IDDM and DN,
and in 192 normoalbuminuric IDDM controls matched for age, sex, and
duration of diabetes. Plasma renin and total renin were measured by
immunoradiometric assays. Genotyping was PCR-based. RESULTS: Plasma renin
was increased in patients with nephropathy (median (range), 26.3
(5.2-243.3) vs 18.3 (4.2-373.5) microU/ml in the normoalbuminuric group,
P<0.0001). Prorenin levels were elevated out of proportion to renin levels
in nephropathic patients (789 (88-5481) vs 302 (36-2226) microU/ml,
P<0.0001). Proliferative retinopathy had an additive effect on plasma
prorenin, but not on renin. DN was associated with a BglI RFLP in the
first intron of the renin gene (bb-genotype: n=106 vs 82 in DN and
normoalbuminuric patients respectively, P=0.037), but not with three other
polymorphisms in the renin gene. A trend for association of higher
prorenin levels with the DN-associated allele of this renin polymorphism
was observed in a subgroup of patients with DN (bb vs Bb+BB, P=0.07).
CONCLUSIONS: The results indicate that in DN there is an increase in both
renin and prorenin levels. A renin gene polymorphism may contribute weakly
to DN. Although speculative, one of the renin gene alleles could lead to
increased renin gene expression, leading to higher renin and prorenin
levels. These may play a role in the pathogenesis of DN
Aliskiren-binding increases the half life of renin and prorenin in rat aortic vascular smooth muscle cells
Renin inhibition with aliskiren has been reported to cause a greater rise in renin than other types of renin-angiotensin system blockade, thereby potentially leading to angiotensin generation or stimulation of the human (pro)renin receptor (h(P)RR). Here we studied whether this rise in renin is attributable to an aliskiren-induced change in the prorenin conformation, allowing its detection in renin assays, or a change in renin/prorenin clearance. We also investigated whether aliskiren affects (pro)renin binding to its receptors, using rat aortic vascular smooth muscle cells (VSMCs) overexpressing the h(P)RR. Methods and Results-A 48-hour incubation with aliskiren at 40C converted the prorenin conformation from "closed" to "open," thus allowing its recognition in active site-directed renin assays. VSMCs accumulated (pro)renin through binding to mannose 6-phosphate receptors (M6PRs) and h(P)RRs. Aliskiren did not affect binding at 40C. At 370C, aliskiren increased (pro)renin accumulation up to 40-fold, and M6PR blockade prevented this. Aliskiren increased the intracellular half life of prorenin 2 to 3 times. Conclusion-Aliskiren allows the detection of prorenin as renin, and decreases renin/prorenin clearance. Both phenomena may contribute to the "renin" surge during aliskiren treatment, but because they depend on aliskiren binding, they will not result in angiotensin generation. Aliskiren does not affect (pro)renin binding to its receptors
Aliskiren accumulates in renin secretory granules and binds plasma prorenin
The vascular effects of aliskiren last longer than expected based on its half life, and this renin inhibitor has been reported to cause a greater renin rise than other renin-angiotensin system blockers. To investigate whether aliskiren accumulation in secretory granules contributes to these phenomena, renin-synthesizing mast cells were incubated with aliskiren, washed, and exposed to forskolin in medium without aliskiren (0.1 to 1000 nmol/L). (Pro)renin concentrations were measured by renin- and prorenin-specific immunoradiometric assays, and renin activity was measured by enzyme-kinetic assay. Without aliskiren, the culture medium predominantly contained prorenin, the cells exclusively stored renin, and forskolin doubled renin release. Aliskiren dose-dependently bound to (pro)renin in the medium and cell lysates and did not alter the effect of forskolin. The aliskiren concentrations required to bind prorenin were 1 to 2 orders of magnitude higher than those needed to bind renin. Blockade of cell lysate renin activity ranged from 27±15% to 79±5%, and these percentages were identical for the renin that was released by forskolin, indicating that they represented the same renin pool, ie, the renin storage granules. Comparison of renin and prorenin measurements in blood samples obtained from human volunteers treated with aliskiren, both before and after prorenin activation, revealed that ≤30% of prorenin was detected in renin-specific assays. In conclusion, aliskiren accumulates in renin granules, thus allowing long-lasting renin-angiotensin system blockade beyond the half-life of this drug. Aliskiren also binds to prorenin. This allows its detection as renin, and might explain, in part, the renin rise during renin inhibition