Recombinant Human Prorenin Research Articles

Abstract 097: Megalin: A Novel Endocytic Receptor for Prorenin and Renin?

Disturbed renal handling of prorenin and renin [(pro)renin] may result in hypertension and renal damage. Megalin is a major endocytic receptor playing an essential role in nutrient reabsorption. Genetic mutations resulting in impaired expression or trafficking of megalin in patients with Dent’s disease or Lowe syndrome caused a 20-40-fold rise of urinary renin levels. Interestingly, the urine of such patients also contained prorenin, which normally is undetectable in urine. Moreover, inhibiting megalin in rodents massively upregulated urinary renin levels. The aim of this study was to investigate (pro)renin binding and internalization by megalin. Immortalized Brown Norway Rat yolk sac cells (BN16) were used, since they abundantly express megalin. To distinguish binding and internalization, cells were incubated at 4°C and 37°C, respectively, with 1000 U/L recombinant human (pro)renin for up to 24 hours. (Pro)renin levels were assessed using an immunoradiometric assay. At 4°C, BN16 cells bound 3 times more prorenin than renin (14184.0±2626.0 vs. 4795.0±986.5 pg/mg of protein, p=0.0155), suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. Mannose-6-phosphate (M6P), an antagonist of the (pro)renin-binding M6P receptor, did not affect cellular (pro)renin uptake. Yet, inhibiting megalin expression using siRNAs reduced (pro)renin binding and internalization by >50%. Furthermore, treating cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin. Exposing prorenin’s prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin is a novel endocytic receptor for (pro)renin which determines urinary (pro)renin levels. Megalin displays selectivity towards prorenin in a prosegment-dependent manner, and this explains why urine normally does not contain prorenin, except under conditions where megalin trafficking is disturbed.

Abstract P235: Renin and Prorenin Uptake by Human Conditionally Immortalized Proximal Tubule Epithelial Cells Involves Megalin and Mannose 6-phosphate Receptor

Renin is filtered by the glomerulus, and subsequently reabsorbed in the proximal tubule in a megalin-dependent manner, as evidenced by a 20-40-fold rise in urinary renin in patients with Dent’s disease or Lowe syndrome, i.e. disorders characterized by defective proximal tubular reabsorption. Remarkably, the reabsorption of filtered prorenin appears to be complete, as it can only be detected in urine of patients with Dent’s disease or Lowe syndrome. To further investigate megalin-mediated renin and prorenin reabsorption, human conditionally immortalized proximal tubule epithelial cells (ciPTEC) were incubated at 37 °C or 4 °C with recombinant human renin or prorenin (1000 U/L), in the absence or presence of 10 mmol/L mannose 6-phosphate (M6P) to block (pro)renin binding and internalization via M6P receptors. Cell lysate (pro)renin levels were measured by immunoradiometric assay both before and after prorenin activation. At 37 °C, cellular renin and prorenin tended to level off at 6 hours, reaching lysate levels of 75±18 and 100±54 pg/mg (mean±SEM), and all prorenin was detected as renin. Incubating ciPTEC at 4 °C diminished the cellular accumulation of renin and prorenin by 90%, and at this temperature prorenin remained in its inactive conformation. M6P blocked renin and prorenin uptake by >80% under all conditions. Importantly, M6P also concentration-dependently blocked the uptake of fluorescently labeled albumin (IC 50 = 3 mmol/L), to the same degree as the megalin inhibitor RAP-GST (IC 50 = 50 μg/mL). Since albumin is internalized in a megalin-dependent manner, these data indicate that M6P, on top of blocking M6P receptors, also interferes with the megalin-dependent uptake process. In conclusion, ciPTEC bind and internalize renin and prorenin in a temperature-dependent manner. This process involves megalin and/or M6P receptors, and results in prorenin activation at 37 °C.

The synthesis, secretion and uptake of prorenin in human amnion.

Very high concentrations of prorenin protein occur in human amniotic fluid and amnion. The source of amniotic fluid prorenin is likely the decidua, as it has the highest levels of prorenin mRNA (REN). Conversely, REN mRNA levels in amnion and chorion are very low. This study aimed to investigate whether decidual prorenin could cross the amnion and accumulate in amniotic fluid. Late gestation amnion was incubated for 24 h in the presence or absence of recombinant human (rh)prorenin. REN mRNA abundance was determined by qPCR and prorenin protein levels in the supernatant and tissue were measured by an ELISA. Prior to incubation only 3/11 amnion samples had REN mRNA but measurable levels of prorenin protein were found (1.4 ng/mg total protein). After 24 h incubation, REN mRNA was found in all explants and levels were significantly increased (P = 0.03) but prorenin protein levels in amnion were unchanged. Prorenin protein levels in the supernatant were, however, increased (P = 0.048). Incubation with (rh)prorenin significantly increased amnion tissue prorenin levels (2.8 ng/mg total protein, P = 0.001); REN mRNA levels were unchanged. Therefore, amnion explants express small amounts of REN and secrete prorenin protein. Prorenin is also taken up by amnion. We postulate that the amniotic renin angiotensin system (RAS) alters pregnancy outcome through effects on gestation length and amniotic fluid volume. Since human amnion can take up and secrete prorenin protein, the activity of both amnion and amniotic fluid RASs can be amplified by prorenin produced by other intrauterine tissues.

Abstract P264: Interaction of Collecting Duct-Derived Prorenin and Soluble Prorenin Receptor Increases Intraluminal Renin Activity and Augments Intratubular Angiotensin II Formation in Ang II-Dependent Hypertensive Rats

Upregulation of collecting duct (CD)-derived renin (CD renin) in angiotensin II (Ang II)-dependent hypertension may provide a pathway for intratubular Ang II formation by acting on angiotensinogen (AGT) delivered from proximal tubule segments. Recently, a prorenin/renin receptor (PRR) has been cloned and shown to enhance renin and prorenin activation. The soluble form of the PRR (sPRR) is augmented in the renal inner medulla of chronic Ang II-infused rats. The present study was performed to determine if renin is secreted into the lumen by the CD cells in chronic Ang II-infused rats and to establish the functional contribution of sPRR to the enhanced renin activity in distal nephron segments. Accordingly, urinary levels of renin ( uRen ) and Ang II ( uAngII ) were measured by RIA in chronic Ang II-infused male Sprague-Dawley rats [80 ng/min, SC minipumps for 14 d, n=10] and sham-operated rats [n=10]. Systolic blood pressure increased in the Ang II rats by Day 5 and continued to increase throughout the study (Day 13; Ang II: 175±10 vs. sham: 116±2 mmHg; p <0.05). Although plasma renin activity (PRA) was suppressed in the Ang II-infused rats, renal medullary renin content was significantly augmented (12,605±1,343 vs. 7,956±765 ng Ang I/h/mg; p <0.05). The excretion of uAngII was also increased (3,813±431 vs. 2,080±361 fmol/day; p <0.05). In addition, renin and prorenin excretion rates increased progressively and were markedly augmented by Day 13 of Ang II infusion [renin (8.6±1.5 vs. 2.8±0.5x10 -6 Enzyme Units Excreted (EUE) /day; prorenin: 15.8 ± 2.8 vs. 2.6 ± 0.7x10 -3 EUE /day, p <0.05). Renin and prorenin protein levels examined by Western Blot in the urine were similarly increased. Importantly, the incubation of urine samples of Ang II-infused rats with recombinant human prorenin showed increased Ang I formation compared to sham-operated rats. In conclusion, in chronic Ang II-infused rats, the presence of sPRR in the urine reflects augmented enzymatic activity of prorenin secreted by the principal cells of the CD, which increase intratubular Ang II de novo formation in the distal nephron segments thus contributing to enhanced sodium reabsorption during Ang II-dependent hypertension.

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 K(D) 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 (r(2)=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.

Molecular evidence of a (pro)renin/ (pro)renin receptor system in human intrauterine tissues in pregnancy and its association with PGHS-2

Prorenin stimulates decidual prostaglandin (PG) production in vitro, the (pro)renin receptor ((P)RR) may mediate this action. The role of prorenin in amnion PG synthesis has not been examined, despite this being the key site of PG synthesis. To determine if (P)RR, prorenin and PGHS-2 are co-localized in gestational tissues and if expression is altered by labour, term amnion, chorion, decidua and placenta were collected during elective caesarean section or after spontaneous labour. Prorenin, (P)RR and PGHS-2 mRNA abundance was determined by real-time RT-PCR. (P)RR protein was examined by immunohistochemistry. The effect of recombinant human (rh) prorenin on PGHS-2 mRNA abundance in amnion explants was determined. Prorenin and (P)RR mRNA were highest in decidua and placenta, respectively. Decidual prorenin, (P)RR and placental (P)RR mRNA abundance decreased with labour. (P)RR protein was present in all gestational tissues. After labour, decidual prorenin was positively correlated with amnion PGHS-2 mRNA and rh-prorenin significantly increased PGHS-2 mRNA abundance in amnion explants. We conclude that the decidua is the principal source of prorenin and is downregulated with labour. All gestational tissues are targets for prorenin. Decidual prorenin may be involved in the labour-associated increase in amnion PGHS-2 abundance via the (P)RR.

118. THE PRORENIN RECEPTOR/PLZF PATHWAY IN HUMAN AMNION

Prorenin, despite being inactive, is the major form of renin found in amniotic fluid and reproductive tissues. Prorenin becomes active if it binds to the novel prorenin receptor (ATP6AP2). The prorenin-ATP6AP2 complex has been found to stimulate translocation of Promyelocytic Zinc Finger (PLZF) protein to the nucleus where it increases expression of the p85α subunit of PI3 kinase (PI3K-p85α) and represses the expression of ATP6AP21. Progesterone and glucocorticoids have also been shown to stimulate PLZF2, 3. We aimed to find out if PLZF and the prorenin-ATP6AP2 pathway interact in human reproductive tissues. Human amnion was cultured for 24 h in media containing vehicle, dexamethasone, amniotic fluid or recombinant human (rh) prorenin. Total RNA was extracted using TRIZol® and converted to cDNA for quantitative real-time PCR using SuperScript III and random hexamers. mRNA abundances for PLZF, PI3K-p85α and ATP6AP2 were calculated relative to Alien RNA using the ΔΔCT method. Our preliminary data show that exposure of amnion explants to dexamethasone upregulates PLZF and PI3K-p85α mRNA but has no effect on ATP6AP2. Culture of amnion explants with amniotic fluid also increases PLZF but does not change PI3K or ATP6AP2. In contrast, culture of amnion explants with (rh) prorenin increases PI3K mRNA but not PLZF or ATP6AP2. As expected, dexamethasone affects PLZF expression, however in amnion there is no interaction with the ATP6AP2 pathway. In addition, we believe we have identified a novel prorenin/ATP6AP2 signalling pathway which acts on PI3K-p85α independent of PLZF. In contrast to these data, amniotic fluid increases PLZF but not PI3K-p85α mRNA levels suggesting that amniotic fluid contains other factors that oppose prorenin and glucocorticoid effects on PI3K-p85α. (1) Schefe, J.H., et al. Circ Res, 2006. 99(12): 1355–1366.(2) Fahnenstich, J., et al. Mol. Hum. Reprod., 2003. 9(10): 611–623.(3) Conquest, A. et al. Fetal and Neonatal Physiology Workshop, 2010. Wellington, New Zealand.

The prorenin induced extracellular signal in HUVECs and HRMCs by the (pro)renin receptor is not blocked by the putative (pro)renin receptor blocker HRP

Objective To determine the mechanism whereby the prorenin activates (pro)renin receptor [(P)RR] on mitogen-activated protein kinases (MAPK) in human umbilical vein endothelial cells (HUVECs) and human renal mesangial cells (HRMCs). To test whether the “handle-region” peptide (HRP) consisting of 10 amino acids from the prorenin prosegment acts as a blocker of (P)RR. Design and method Immunocytochemistry staining was used to evaluate the presence of (P)RR in HUVECs. The cells were treated with recombinant human prorenin after the blockage of AT1 and AT2 by olmesartan and PD123319.

The effect of the (pro)renin receptor on the activation of extracellular signal-regulated kinases 1 and 2 on human renal mesangial cells

Objectives: To investigate whether human prorenin can provoke the (pro)renin receptor [(P)RR] leading to the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and whether the putative (pro)renin receptor blocker “handle-region” peptide (HRP) inhibits this pathway in cultured human renal mesangial cells (HRMCs). Methods: HRMCs were cultured in vitro. HRMCs were pretreated with the AT1 blocker olmesartan and AT2 blocker PD123319 blocker for 30 min, then HRMCs were stimulated by prorenin, PD98059 inhibitor of the phosphorylation of ERK1/2 and HRP respectively. Western blot was used to test the concentration of p-ERK1/2. The concentration of transfer growth factor-β1 (TGF-β1) was measured by the ELASA method. The mRNA of TGFβ1 was measured by the RT-PCR method. Results: We found that recombinant human prorenin induced the activation of (P)RR in cultured HRMCs, and in turn, increased the phosphorylated ERK1/2. Recombinant humanprorenin induced a rapid phosphorylation of ERK1/2 and pREK1/2 which increased in a timeand dose-dependent way, and the prorenin levels of TGF-β1 increased significantly. We also found that an ERK inhibitor PD98059 significantly decreased the phosphorylated ERK1/2 and then down regulated the TGF-β1. The putative blocker HRP of (P)RR which consisted of the handle-region decoy peptide of prorenin inhibits neither the phosphorylation of ERK1/2 nor the increase of TGFβ1. Conclusion: Prorenin induced a long-lasting ERK1/2 phosphorylation in a timeand concentration-dependent manner in HRMCs. The signal transduction is different from that which is provoked by Ang II. TGF-β1 increased obviously accompanied by the ERK1/2 phosphorylation. PD98059 decreased the production of TGF-β1 induced by (P)RR. HRP affects neither the signaling in HRMCs nor the TGF-β1.

(Pro)Renin Receptor-Mediated Activation of Mitogen-Activated Protein Kinases in Human Vascular Smooth Muscle Cells

Blockade of (pro)renin receptor has benefits in diabetic angiotensin II type-1a-receptor-deficient mice, suggesting the importance of (pro)renin receptor-mediated intracellular signals. To determine the mechanism whereby the human (pro)renin receptor activates mitogen-activated protein kinases in human vascular smooth muscle cells (hVSMC), we treated the cells with recombinant human prorenin. Prorenin enhanced hVSMC proliferation and activated extracellular-signal-related protein kinase (ERK) in a dose- and time-dependent manner but did not influence activation of p38 or c-Jun NH(2)-terminal kinase. The activated ERK level was reduced to the control level by the tyrosine kinase inhibitor genistein, and the MEK inhibitor U0126 markedly reduced the activated ERK level to the control level, whereas the level of activated ERK was unaffected by the angiotensin-converting enzyme inhibitor imidaprilat or the angiotensin II receptor blocker candesartan. A human (pro)renin receptor was present in hVSMCs, and its knockdown with small interfering RNA (siRNA) significantly inhibited the prorenin-induced ERK activation. These results suggest that prorenin stimulates ERK phosphorylation in hVSMCs through the receptor-mediated activation of tyrosine kinase and subsequently MEK, independently of the generation of angiotensin II or the activation of its receptor. The (pro)renin receptor-mediated ERK signal transduction is thus a possible new therapeutic target for preventing vascular complications.

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

Human Prorenin Has “Gate and Handle” Regions for Its Non-proteolytic Activation

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.

Cardiomyocytes Bind and Activate Native Human Prorenin

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

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.

Cardiomyocytes Bind and Activate Renal But not Non-Renal Human Prorenin.

P10 Cardiomyocytes bind and internalize recombinant human prorenin (rhPR) and renin via mannose 6-phosphate (M6P) receptors (van Kesteren et al., Hypertension 1997). PR binding is followed by intracellular activation. Here we investigated 1) whether cardiomyocytes bind native human (pro)renin ((P)R) from various sources and 2) whether PR binding is followed by activation. Myocytes were isolated from Wistar rat pups by enzymatic dissociation and grown to confluence (6 x 10 5 cells in 3.8 cm 2 wells). Cells were incubated with rhPR, human plasma (obtained from captopril-treated patients (CAP), patients with renal artery stenosis (RAS) and anephric subjects (Nx)), or human amniotic fluid (HAF), diluted in DMEM (final volume 0.4 mL), for 4 hrs at 37°C, either with or without 10 mM M6P. Cell-associated R and PR, the latter following prior activation by plasmin, were determined by enzyme-kinetic assay. Results: See Table. Intracellular PR activation is reflected by the higher R/PR ratio in the cell lysates as compared to the medium. Conclusions: Cardiomyocytes bind and activate native human PR from renal (CAP & RAS), but not from non-renal (Nx & HAF) sources in a M6P- dependent manner. These data suggest that renal PR is meant to be sequestered by nonrenin-producing tissues in order to contribute to local angiotensin production, whereas non-renal PR functions at its production site only.

Cardiomyocytes Bind and Activate Renal But not Non-Renal Human Prorenin.

P10 Cardiomyocytes bind and internalize recombinant human prorenin (rhPR) and renin via mannose 6-phosphate (M6P) receptors (van Kesteren et al., Hypertension 1997). PR binding is followed by intracellular activation. Here we investigated 1) whether cardiomyocytes bind native human (pro)renin ((P)R) from various sources and 2) whether PR binding is followed by activation. Myocytes were isolated from Wistar rat pups by enzymatic dissociation and grown to confluence (6 x 10 5 cells in 3.8 cm 2 wells). Cells were incubated with rhPR, human plasma (obtained from captopril-treated patients (CAP), patients with renal artery stenosis (RAS) and anephric subjects (Nx)), or human amniotic fluid (HAF), diluted in DMEM (final volume 0.4 mL), for 4 hrs at 37°C, either with or without 10 mM M6P. Cell-associated R and PR, the latter following prior activation by plasmin, were determined by enzyme-kinetic assay. Results: See Table. Intracellular PR activation is reflected by the higher R/PR ratio in the cell lysates as compared to the medium. Conclusions: Cardiomyocytes bind and activate native human PR from renal (CAP & RAS), but not from non-renal (Nx & HAF) sources in a M6P- dependent manner. These data suggest that renal PR is meant to be sequestered by nonrenin-producing tissues in order to contribute to local angiotensin production, whereas non-renal PR functions at its production site only.

Uptake and proteolytic activation of prorenin by cultured human endothelial cells.

To investigate the mechanisms of vascular uptake of prorenin and renin and to explore the possibility of vascular activation of prorenin. Human umbilical vein endothelial cells (HUVECs) cultured in a chemically defined medium were incubated with recombinant human prorenin or renin in the presence or absence of putative inhibitors of renin internalization. Cell surface-bound and internalized prorenin or renin were separated by the acid-wash method and were quantified by enzyme-kinetic assays. The activation of prorenin was also monitored by a direct immunoradiometric assay (IRMA) with use of a monoclonal antibody directed against the -p24-Arg to -1p-Arg C-terminal propeptide sequence of prorenin. Prorenin and renin were internalized at 37 degrees C in a dose-dependent manner; with 1000 microU prorenin/ml medium, the quantity of cell-associated prorenin after 3 h of incubation was 9.3 +/- 1.0 microU/4 x 10(5) cells, and with 75,000 microU/ml medium it was 670 +/- 75 microU/4 x 10(5) cells (mean +/- SD; n = 5). Results for renin were similar. Prorenin that had been treated with endoglycosidase H to remove N-linked oligosaccharides was not internalized. Addition of mannose 6-phosphate (M-6-P) to the medium caused a dose-dependent inhibition of renin and prorenin internalization. Fifty per cent inhibition was observed at 70 micromol/M-6-P, whereas mannose 1-phosphate, glucose 6-phosphate and alpha-methylmannoside at this concentration had no effect Ammonium chloride (50 mmol/l) and monensin (10 micromol/l) also inhibited internalization. Prorenin was activated by HUVECs, and cell-activated prorenin was only found in the internalized fraction, whereas the surface-bound prorenin remained inactive. Thus, it appears that the activation of prorenin took place at the time of its internalization or thereafter. The results of the prorenin IRMA indicated that activation was associated with proteolytic cleavage of the propeptide. Our findings provide evidence for M-6-P receptor-dependent endocytosis of (pro)renin and proteolytic prorenin activation by vascular endothelial cells.

Non-proteolytic activation of human prorenin by anti-prorenin prosegment (pf#1: 1P-15P) antiserum.

Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L1PPTDTTTFKRIFLKR15P) antiserum at 4 degrees C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L1PPTDTTTF8P as well as L1PPTDTTTFKRIFLKR15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The Km and Vmax values of the activated prorenin were 0.2 microM and 23.7 micrograms Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.

Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells.

The binding and internalization of recombinant human renin and prorenin (2500 microU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37 degrees C and 4 degrees C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37 degrees C, after incubation at 4 degrees C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37 degrees C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor-dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.

Enzymatic characterization of purified recombinant human renin.

Renin is a highly specific aspartyl protease of the renin-angiotensin system initially synthesized as preprorenin. Recombinant human prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10-15 mg of recombinant human renin was secreted in the supernatant from each cell factory. Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity. This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I.mg protein-1.h-1 was used for kinetic analysis. The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1-Asn14, at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Km and kcat values for the peptidic substrate (13.3 microM and 8.1 s-1, respectively), when compared with values for the natural substrate (2.04 microM and 1.41 s-1), the catalytic efficiency (0.69 microM-1.s-1) of the enzyme for both substrates was the same. However, the kcat/Km value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5. The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin.