Activation Of Prorenin Research Articles

Abstract 043: Prorenin Reduces Spontaneous Firing Rate In Cells Expressing Tyrosine Hydroxylase In The Hypothalamic Paraventricular Nucleus

The (pro)renin receptor (PRR) plays essential roles in cardiovascular and metabolic physiology. The tyrosine hydroxylase (TH) neurons in the paraventricular nucleus of the hypothalamus (PVN) have recently emerged as a key metabolic regulator that play functional roles in the regulation of the energy expenditure and metabolism. Using RNAScope in situ hybridization, we found that 95% of TH-containing neurons in the PVN (n=872 cells/3 mice) express the PRR. In this study, we aimed to examine the effects of PRR activation on the firing activity of the TH-containing neurons in the PVN (TH PVN ). We hypothesized that prorenin activation of the PRR increases firing activity of the TH PVN neuron. To test this hypothesis, we performed electrophysiological recordings in loose cell-attached voltage-clamp configuration in PVN slices from mice expressing TdTomato reporter for TH neurons (TH-cre-TdTomato mice). These experiments were carried out to isolate and record spontaneous firing rate (SFR). We abolished all the synaptic transmission by adding a GABA A receptor antagonist (picrotoxin, 50 μM), an antagonist of AMPA/kainate receptors (CNQX, 5 μM), and an NMDA receptor antagonist (D-APV, 50 μM) throughout the recording. The cells were discarded if the access resistance was >15 MΩ or fluctuated more than 15%. At the end of the experiments, tetrodotoxin (1 μM) was added to abolish and confirm neuronal activity. The SFR in TH PVN neurons was significantly decreased after application of prorenin (PR) (4.9 ± 0.87 and 3.2 ± 1.07 Hz, before and after PR, respectively; n = 10, p = 0.006, paired t -test) indicating that PR reduces spontaneous firing activity of the TH PVN neurons. To determine if this effect was due to the prorenin activation of the PRR, we recorded SFR in the presence of PRR antagonist (PRO20). There was no difference in the SFR (5.8 ± 1.58 Hz and 5.9 ± 1.7 Hz, n = 5, p = 0.903, paired t -test) in the TH PVN neurons between PRO20 and PRO20+PR group respectively suggesting that PRO20 blocks the effect of PR in reducing SFR in TH PVN neurons. Our results showed that the prorenin acts on the PRR to reduce the SFR of the TH PVN neurons. We conclude that PRR signaling in the TH PVN neurons might play a role in cardiovascular and metabolic regulation by the modulating the TH PVN neuronal plasticity.

Deletion of Prorenin Receptor in the Rostral Ventrolateral Medulla Results in Biphasic and Sex‐Dependent Pressor Responses in Deoxycorticosterone Acetate‐salt Hypertension

In models of low renin hypertension such as the deoxycorticosterone acetate (DOCA)‐salt model, the local generation of angiotensin II in the brain mediates the increase in blood pressure. The prorenin receptor (PRR) might play a potential role in the local activation of prorenin in the brain. We have new evidence that prorenin and PRR are co‐expressed in the proximity to the rostral ventrolateral region of the medulla oblongata (RVL). Therefore, we hypothesized that selective ablation of PRR targeting the RVL attenuates responses to DOCA‐salt in mice. PRR ablation was targeted to the RVL by stereotactic microinjections of adeno‐associated virus (AAV) expressing Cre recombinase in PRR‐flox mice (PRRRVL‐KO). AAV mCherry was used as a control virus (WT). Pressor response to L‐glutamate in the injection site served as confirmatory stereotactic target hit. During the first week of DOCA‐salt treatment, RVL‐targeted ablation of PRR resulted in lower pressor responses to DOCA‐salt in females (WT=115±3 vs KO=104±4 mmHg; p<0.05; n=8), but not males. However, after week 2 on DOCA‐salt treatment, female PRRRVL‐KO unexpectedly exhibited an exaggerated increase in systolic blood pressure (WT=149±3 vs KO=163±3 mmHg; p=0.004; n=8) and this was accompanied by a trending increase in urinary norepinephrine (WT=177±28 vs KO=270±38 ng/ml; p=0.07; n=7‐8). Next, female mice were challenged with an intraperitoneal hypertonic saline injection equivalent to 10% of their body weight followed by 4 hours of urine collection at baseline and on DOCA‐salt treatment. Urinary sodium excretion in female PRRRVL‐KO was significantly lower when compared to WT in both conditions (p<0.05). Finally, using nuclear magnetic resonance/bioimpedance spectroscopy we assessed body fluid compartmentalization. We observed no difference in total body fluid among groups at any time point, but there was a significant mobilization of extracellular fluid to the intracellular compartment in PRRRVL‐KO. Our data indicate that the role of PRR in the RVL is sex‐dependent and biphasic. That is, PRR contributes to the pressor response during the initial stage of DOCA‐salt hypertension in females, presumably by facilitating the generation of angiotensin peptides in the RVL, while it plays a protective role by promoting renal sodium excretion and preventing blood pressure elevation during the maintenance stage of DOCA‐salt hypertension. This study also suggests that distinct PRR expressing cell populations within the RVL might elicit diverging physiological functions.

The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5–10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

Effect of prorenin peptide vaccine on the early phase of diabetic retinopathy in a murine model of type 2 diabetes.

Prorenin is viewed as an ideal target molecule in the prevention of diabetic retinopathy. However, no drugs are available for inhibiting activation of prorenin. Here, we tested the effect of a prorenin peptide vaccine (VP) in the retina of a murine model of type 2 diabetes (T2D). To choose the optimal vaccine, we selected three different epitopes of the prorenin prosegment (E1, E2, and E3) and conjugated them to keyhole limpet hemocyanin (KLH). We injected C57BL/6J mice twice with KLH only (as a control vaccine), E1 conjugated with KLH (E1-KLH), E2-KLH, or E3-KLH and compared antibody titers. E2-KLH showed the highest antibody titer and specific immunoreactivity of anti-sera against prorenin, so we used E2-KLH as VP. Then, we administered injections to the non-diabetic db/m and diabetic db/db mice, as follows: db/m + KLH, db/db + KLH, and db/db + VP. Retinal blood flow measurement with laser speckle flowgraphy showed that the impaired retinal circulation response to both flicker light and systemic hyperoxia in db/db mice improved with VP. Furthermore, the prolonged implicit time of b-wave and oscillatory potentials in electroretinography was prevented, and immunohistochemical analysis showed reduced microglial activation, gliosis, and vascular leakage. The enzyme-linked immunosorbent spot assay confirmed vaccinated mice had no auto-immune response against prorenin itself. The present data suggest that vaccination against prorenin is an effective and safe measure against the early pathological changes of diabetic retinopathy in T2D.

Abstract P198: Biphasic And Sex-dependent Roles Of The Prorenin Receptor In The Rostral Ventrolateral Nucleus In Mice Subjected To Deoxycorticosterone Acetate-salt Hypertension

The brain renin angiotensin system (RAS) regulates blood pressure (BP) and autonomic function. However, it remains unclear how and where angiotensin II (Ang II) is generated in conditions eliciting brain RAS overactivation including deoxycorticosterone acetate (DOCA)-salt hypertension (HT). In several tissues, the activation of prorenin requires its binding to the prorenin receptor (PRR). New evidence from this study indicates that prorenin and PRR are co-expressed in the proximity to the rostral ventrolateral nucleus (RVL), an anatomical brain region that controls sympathetic nerve activity. Therefore, we hypothesized that selective ablation of PRR targeting the RVL attenuates BP increase due to DOCA-salt. PRR ablation was targeted to the RVL by stereotactic microinjections of adeno-associated virus (AAV) expressing Cre recombinase-mCherry in PRR-flox mice (PRR RVL-KO ). AAV mCherry was used as control virus (WT). A pressor response to L-glutamate in the injection site served as confirmatory stereotactic target hit. RVL-targeted ablation of PRR resulted in lower BP responses to DOCA-salt in females (WT=115±3 vs KO=104±4 mmHg; p <0.05; n=8), but not males (n=5-8), only during the first 3 days of DOCA-salt treatment. However, at day 13 of DOCA-salt treatment, female PRR RVL-KO unexpectedly exhibited exaggerated increase in systolic BP (WT=149±3 vs KO=163±3 mmHg; p =0.004; n=8) and pulse pressure (WT=31±4 vs KO=45±4 mmHg; p =0.02; n=8) when compared to control. Next, mice were challenged with an intraperitoneal hypertonic saline injection equivalent to 10% of their body weight followed by 4 hours of urine collection. Urinary sodium excretion in female PRR RVL-KO was significantly lower when compared to WT ( p <0.05). These data indicate that the role of PRR in the RVL is sex-dependent and biphasic. That is, PRR contributes to the pressor response during the initial stage of DOCA-salt HT in females, presumably by facilitating the generation of angiotensin peptides in the RVL, while it plays a protective role by promoting renal sodium excretion and preventing elevation of systolic BP during the maintenance stage of DOCA-salt HT. This study suggests that distinct PRR expressing cell populations might elicit diverging physiological functions within the RVL.

High glucose induces trafficking of prorenin receptor and stimulates profibrotic factors in the collecting duct

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague–Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

Effect of HRP Upon the Expression and Intracellular Signaling of PRR in Cardiovascular and Renal Systems in Preeclamptic Rats Offspring

Introduction Preeclampsia (PE) is a condition that affects both, the mother and the child, conditioning in both a greater susceptibility to develop cardiovascular diseases in future stages. It has been studied that prorenin renin receptor (PRR) has a broad participation in the cardiovascular system of PE products, it seems to play an important role in the development of future conditions, such as arterial hypertension, involving the renin angiotensin aldosterone system (RAS). RAS hyperactivity has been implicated as a cardiovascular risk factor in patients who come from a pregnancy complicated by preeclampsia. Mechanisms mediating fetal programming of adult arterial hypertension are numerous, including vascular dysfunction and reduction of the density of the arterioles and capillaries. Handle Region Peptide (HRP) is a decoy peptide that can inhibit the binding of prorrenin to the PRR receptor. HRP mimics the loop region and therefore competitively binds to the receptor, thus preventing receptor-mediated activation of prorenin and reducing its activity in tissue. Objective To evaluate the HRP blockade upon the PRR receptor and its transduction pathways: WNT and MAPK in cardiovascular and renal tissue in offspring from a pre-eclampsia model. Material and methods Male and female offspring from PE and healthy pregnant wistar rats, with 30 and 120 days of age, where set up in groups with or without HRP treatment (0.2 mg/Kg, subcutaneously for 7 days before sacrifice). We measured blood pressure by tail-cuff method. At the end of the treatment the expression of PRR, DVL1, beta-catenin, PKC-alpha were measured by immunoblot and RT-PCR in cardiovascular and renal tissue. Results Offspring from PE rats showed sustained high blood pressure, both male and female at 30 and 120 days. HRP treatment reverted partially this increase. All PE-derived groups showed an increase in PRR expression in aorta, heart and kidney. WNT and MAPK pathways also seemed to be activated in PE-derived animals, as shown by β-catenin, DVL-1 and PKCa increased expression. These findings are important because WNT signaling pathway is associated with kidney diseases and cardiac hypertrophy. Besides, this work shows that PRR participates in high blood pressure development in offspring derived from PE pregnancies.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the “handle region peptide” from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

The evolving complexity of the collecting duct renin-angiotensin system in hypertension.

The intrarenal renin-angiotensin system is critical for the regulation of tubule sodium reabsorption, renal haemodynamics and blood pressure. The excretion of renin in urine can result from its increased filtration, the inhibition of renin reabsorption by megalin in the proximal tubule, or its secretion by the principal cells of the collecting duct. Modest increases in circulating or intrarenal angiotensin II (ANGII) stimulate the synthesis and secretion of angiotensinogen in the proximal tubule, which provides sufficient substrate for collecting duct-derived renin to form angiotensin I (ANGI). In models of ANGII-dependent hypertension, ANGII suppresses plasma renin, suggesting that urinary renin is not likely to be the result of increased filtered load. In the collecting duct, ANGII stimulates the synthesis and secretion of prorenin and renin through the activation of ANGII type 1 receptor (AT1R) expressed primarily by principal cells. The stimulation of collecting duct-derived renin is enhanced by paracrine factors including vasopressin, prostaglandin E2 and bradykinin. Furthermore, binding of prorenin and renin to the prorenin receptor in the collecting duct evokes a number of responses, including the non-proteolytic enzymatic activation of prorenin to produce ANGI from proximal tubule-derived angiotensinogen, which is then converted into ANGII by luminal angiotensin-converting enzyme; stimulation of the epithelial sodium channel (ENaC) in principal cells; and activation of intracellular pathways linked to the upregulation of cyclooxygenase 2 and profibrotic genes. These findings suggest that dysregulation of the renin-angiotensin system in the collecting duct contributes to the development of hypertension by enhancing sodium reabsorption and the progression of kidney injury.

ASSOCIATION BETWEEN CARDIAC (PRO)RENIN RECEPTOR EXPRESSION AND MYOCARDIAL HYPERTROPHY IN HUMAN

Objective: The (pro)renin receptor [(P)RR] is expressed in vital organs throughout the body, with the highest expression level in the heart, and is thought to regulate the tissue renin-angiotensin system (RAS) through the non-proteolytic activation of prorenin. (P)RR is cleaved by processing enzymes to generate soluble (s)(P)RR, which is secreted into the extracellular space, and blood concentration of s(P)RR is considered to reflect the expression level of tissue RAS. Although it has been suggested that increased expression of (P)RR may be involved in cardiac pathophysiology in experimental studies, the significance of (P)RR expression in human heart remains unclear. Therefore, in the present studies, we investigated association between cardiac (P)RR expression and myocardial hypertrophy in human. Design and method: Study (1): We measured serum s(P)RR concentration and assessed the relationships between serum s(P)RR level and background factors including left ventricular mass index (LVMI) in 135 patients (57 males) of our department. Study (2): We performed immunostaining for (P)RR and atrial natriuretic peptide (ANP) and α-skeletal actin (SKA), markers for myocardial hypertrophy, in left ventricle specimens obtained from 13 autopsy cases. The expression level of (P)RR, ANP, or SKA and cardiomyocyte area were analyzed using an image processing software (ImageJ) and expression level of (P)RR was compared with cardiomyocyte area and expression level of ANP or SKA. Results: Study (1): The patients’ age was 57 (44–67) y.o., serum s(P)RR concentration was 22.8 (21.1–27.7) ng/ml, eGFR was 74.4 (59.4–91.2) ml/min/1.73 m2, and LVMI was 79 (66–90) g/ m2. Serum s(P)RR level was significantly positively correlated with LVMI (β = 0.238, p < 0.01) independent of other related factors. Study (2): (P)RR immunostaining was observed in cytoplasm of cardiomyocytes. The expression level of (P)RR was significantly positively correlated with cardiomyocyte area (ρ = 0.386, p < 0.001) and expression level of ANP (ρ = 0.782, p < 0.001) or SKA (ρ = 0.373, p < 0.001). Conclusions: These data suggested that cardiac (P)RR expression may be associated with myocardial hypertrophy in human. Further studies are needed to determine the underlying mechanism of this association.

Intrarenal Bradykinin (BK) is Decreased in Mice with Prorenin Receptor (PRR) Deficiency in the Collecting Duct

The renin‐angiotensin system (RAS) and kallikrein‐kinin system (KKS) play a role in multiple physiological and pathological conditions, including the regulation of blood pressure (BP) and sodium reabsorption. The prorenin receptor (PRR), a key component of the RAS, is involved in the control of epithelial sodium channel (ENaC) and BP. Recently, our group reported that bradykinin (BK)/bradykinin type 2 receptor (B2R) activation stimulates the synthesis and release of prorenin/renin in collecting duct (M‐1) cells via PKC and nitric oxide (NO) pathways. We further demonstrated that mice with deficiency of PRR in the collecting ducts (CDPRR‐KO) exhibited attenuated BP and ENaC activity due to decreased amount of renin and angiotensin (Ang) II in the distal nephron during chronic Ang II‐induced hypertension. Taking together these data suggest that PRR is necessary for the activation of prorenin in the distal nephron segments. However, whether intrarenal BK contributes to the decreased levels of distal tubular prorenin/renin in CDPRR‐KO mouse model is unknown. To test the hypothesis that decreased intrarenal BK leads to diminished levels of renin in the collecting duct of CDPRR‐KO mice, we used two groups of mice (N=5–7), including: 1)CDPRR‐KO and Wild type (WT). Systolic blood pressure (SBP) measured by telemetry was decreased in KO compared with WT mice (112±2 vs.123±3 mmHg; P<0.05). The metabolic parameters: body weight, food intake, and water consumption did not show differences between CDPRR‐KO and WT mice. Urine amount of renin was decreased in KO mice (11.5±3 vs. 30.5±11 pg/mL; P<0.05). Plasma levels of BK were quantified by ELISA and resulted similar between both groups of mice (54±4 vs. 51±2 ng/mL; P=0.54). In contrast, urine levels of BK, measured as reflection of intrarenal BK, were significantly decreased in KO mice compared with WT (2.6±0.4 vs. 6.5±0.7 ng/mL; P<0.001). It is known that angiotensin converting enzyme (ACE) is responsible for BK degradation within the kidney. To further examine whether intrarenal ACE explain the differences in intrarenal BK, we quantified ACE activity using a fluoregenic peptide (Abz‐FRK(Dnp)P) in the presence or absence of ACE inhibition with Lisinopril. ACE activity did not differ between CDPRR‐KO and WT mice (5.1±0.2 vs. 4.2±0.5 AU/min/mg prot; P=0.340). In conclusion, these data indicate that intrarenal BK is decreased in CDPRR‐KO mice. Decreased levels of intrarenal BK explain, at least in part, the decreased levels of distal tubular prorenin/renin and ENaC activity in CDPRR‐KO mouse model. The mechanism by which BK is decreased in the kidney of CDPRR‐KO mice is not clear, but it could be a compensatory response in this model. Efforts will be done toward defining the role of PRR in the interactions between the RAS and the KKS and its actual contribution to the regulation of blood pressure.Support or Funding InformationNIDDK (DK‐104375) and LSU Health Sciences Center/Summer Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Do prorenin-synthesizing cells release active, 'open' prorenin?

The function of prorenin, the inactive precursor of renin, remains unclear after many decades of research. The discovery of a (pro)renin receptor suggested that prorenin, by binding to this receptor, would become active, that is, obtain an 'open' conformation. However, the receptor only interacted with prorenin at levels that were many orders of magnitude above its normal levels, making such interaction in-vivo unlikely. Prorenin occurs in two conformations, an open, active form, and a closed, inactive form. Under physiological conditions (pH 7.4, 37 °C), virtually all prorenin occurs in the closed conformation. This study investigated to what degree prorenin-synthesizing cells release prorenin in an open conformation. Renin plus prorenin-synthesizing human mast cells, and prorenin-synthesizing HEK293 cells (transfected with the mammalian expression vector pRhR1100, containing human prorenin) and human decidua cells were incubated with the renin inhibitor VTP-27999. This inhibitor will trap open prorenin, as after VTP-27999 binding, prorenin can no longer return to its closed conformation, thus allowing its detection in a renin immunoradiometric assay. No evidence for the release of open prorenin was found. Moreover, incubating decidua cells with angiotensinogen yielded low angiotensin levels, corresponding with the activity of ≈1% of prorenin in the medium, that is, the amount of open prorenin expected based upon the equilibrium between open and closed prorenin under physiological conditions. Our study does not reveal evidence for the release of open, active prorenin by prorenin-synthesizing cells, at least under cell culture conditions. This argues against prorenin activity at the site of its release.

Expression of (Pro)renin Receptor During Rapamycin-Induced Erythropoiesis in K562 Erythroleukemia Cells and Its Possible Dual Actions on Erythropoiesis.

(Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, is expressed in erythroblastic cells. (P)RR has multiple biological actions: prorenin activation, stimulation of the intracellular signaling including extracellular signal-regulated kinases, and functional complex formation with vacuolar H+-ATPase (v-ATPase). However, the functional implication of (P)RR in erythroblast cells has not been clarified. The aim of the present study was to clarify changes of (P)RR expression during erythropoiesis and a role of (P)RR in the heme synthesis. (P)RR expression was studied during rapamycin-induced erythropoiesis in a human erythroleukemia cell line, K562. Treatment with rapamycin (100 nM) for 48 hours significantly increased %number of hemoglobin-producing cells, γ-globin mRNA levels, erythroid specific 5-aminolevulinate synthase (ALAS2) mRNA levels, and heme content in K562 cells. Both (P)RR protein and mRNA levels increased about 1.4-fold during rapamycin-induced erythropoiesis. Suppression of (P)RR expression by (P)RR-specific small interference RNA increased ALAS2 mRNA levels about 1.6-fold in K562 cells, compared to control using scramble RNA, suggesting that (P)RR may down-regulate ALAS2 expression. By contrast, treatment with bafilomycin A1, an inhibitor of v-ATPase, decreased greatly % number of hemoglobin-producing cells and heme content in K562 cells, indicating that the v-ATPase function is essential for hemoglobinization and erythropoiesis. Treatment with bafilomycin A1 increased (P)RR protein and mRNA levels. In conclusion, we propose that (P)RR has dual actions on erythropoiesis: the promotion of erythropoiesis via v-ATPase function and the down-regulation of ALAS2 mRNA expression. Thus, (P)RR may contribute to the homeostatic control of erythropoiesis.

Abstract P278: On the Origin of Brain Renin

A renin-angiotensin system (RAS) in the brain is believed to contribute to blood pressure regulation. Given the presence of the blood-brain barrier, brain RAS activity most likely depends on synthesis of (pro)renin in the brain. In support of this concept, an intracellular, non-secreted form of renin has been described in the brain, depending on an alternative transcript of the renin gene. To what degree this intracellular truncated prorenin truly displays angiotensin (Ang) I-generating activity (AGA) is unknown. In the present study, we set out to quantify brain (pro)renin, both before and after buffer perfusion of the brain, in normal mice, renin knockout (KO) mice, DOCA-salt-treated mice (which have been reported to display brain RAS activation), and angiotensin II-infused mice. Brain nuclei were homogenized and incubated with excess angiotensinogen to detect AGA, both before and after acid activation of prorenin, with or without the renin inhibitor aliskiren to correct for non-renin-mediated AGA. Renin-dependent (i.e., aliskiren-inhibitable) AGA was readily detectable in brain nuclei, the highest AGA being present in brainstem (>thalamus ≈cerebellum ≈striatum ≈midbrain >hippocampus ≈cortex). Brain AGA increased non-significantly after prorenin activation, suggesting that brain prorenin levels are low or absent. Buffer perfusion reduced AGA in all brain areas by >60%. Plasma renin (expressed per mL plasma) was 40-800 x higher than brain renin (expressed per g tissue). Plasma prorenin levels resembled plasma renin levels. AGA was undetectable in plasma and brain of renin KO mice, both before and after prorenin activation. DOCA-salt and Ang II suppressed plasma renin, and parallel decreases were observed for brain renin. In conclusion, brain renin levels (per g tissue) correspond with the amount of renin present in 1-20 μL blood plasma. Brain renin disappears after buffer perfusion, and varies in association with plasma renin. This indicates that renin detected in brain nuclei represents plasma renin and/or locally activated plasma prorenin. DOCA-salt exposure does not selectively increase brain renin expression.

Patiromer treatment of hyperkalemic resistant hypertension patients with diabetic kidney disease on RAASi normalizes serum potassium and reducing discontinuations secondary to hyperkalemia over 1 year (AMETHYST - DN)

to display brain RAS activation), and Ang II-infused mice. Brain nuclei were homogenized and incubated with excess angiotensinogen to detect AGA, both before and after acid activation of prorenin, with or without the renin inhibitor aliskiren to correct for non-renin-mediated AGA. Renin-dependent (i.e., aliskiren-inhibitable) AGA was readily detectable in brain nuclei, the highest AGA being present in brainstem (> thalamus 1⁄4 cerebellum 1⁄4 striatum 1⁄4 midbrain > hippocampus 1⁄4 cortex). Brain AGA increased non-significantly after prorenin activation, suggesting that brain prorenin levels are low or absent. Buffer perfusion reduced AGA in all brain areas by >60%. Plasma renin (expressed per mL plasma) was 40-800 x higher than brain renin (expressed per g tissue). Plasma prorenin levels resembled plasma renin levels. AGA was undetectable in plasma and brain of renin KO mice, both before and after prorenin activation. DOCA-salt and Ang II suppressed plasma renin, and parallel decreases were observed for brain renin. In summary, brain renin levels (per g tissue) correspond with the amount of renin present in 1-20 mL blood plasma. Brain renin disappears after buffer perfusion, and varies in association with plasma renin. These data indicate that renin detected in brain nuclei represents plasma renin and/or locally activated plasma prorenin. DOCA-salt exposure does not selectively increase brain renin expression.

Abstract 095: Human (Pro)renin Receptor Activation Induces an Angiotensin II-Independent Pressor Response Mediated by NADPH Oxidase 4 Activity

The binding of prorenin to the (pro)renin receptor (PRR) induces non-proteolytic activation of prorenin and generation of angiotensin II (Ang II). PRR activation can also induce Ang II-independent signaling pathways. However, whether Ang II-independent signaling pathways are critical for blood pressure (BP) regulation is not known. To address this question, we created transgenic mice that overexpress the human PRR (hPRR) selectively in neurons (Syn-hPRR). Activated human prorenin (hPRO) cannot cleave endogenous mouse angiotensinogen to generate Ang II. Therefore, administration of hPRO to Syn-hPRR mice can be used to examine Ang II-independent PRR signaling in BP regulation. Intracerebroventricular (ICV) infusion of hPRO increases BP in Syn-hPRR mice (ΔMAP 23 ± 4.6, n = 4) but has no effect on wildtype (WT) mice (ΔMAP 2 ± 0.8, n = 6). The hPRO-induced pressor response in Syn-hPRR mice is unaffected by co-infusion with the Ang II type 1 receptor blocker losartan (ΔMAP 19 ± 5.2, n = 8), suggesting that the response is independent of Ang II. Interestingly, co-infusion with an inhibitor of the reactive oxygen species-generating enzyme NADPH oxidase (NOX), diphenyleneiodonium, nearly abolishes the hPRO-induced pressor response in Syn-hPRR mice (ΔMAP 4.7 ± 1.0, n = 4), indicating that NOX activity is required. Additionally, we find that basal NOX activity is enhanced in the Syn-hPRR hypothalamus relative to WT mice (1.4 fold change). We next examined which NOX isoform is responsible for the hPRO-induced pressor response and enhanced activity. NOX4 mRNA levels are greater (2.7 ± 0.6 fold change), but NOX1 (1.2 ± 0.3 fold change) and NOX2 (1.2 ± 0.3 fold change) mRNA levels are not different, in the hypothalamus of Syn-hPRR compared to WT mice (n = 3). Adenovirus-mediated delivery of NOX2, NOX4, or a scrambled sequence shRNA was ICV injected in Syn-hPRR mice. After 7 days, we found that treatment with NOX2 (ΔMAP 20 ± 5.2) or scrambled (ΔMAP 23 ± 3.2) shRNA had no effect on the hPRO-induced pressor response (n = 5). However, the hPRO-induced increase in BP is attenuated in Syn-hPRR mice injected with NOX4 shRNA (ΔMAP 5.9 ± 2.8). Together, these data indicate that NOX4 mediates the Ang II-independent pressor response to activation of the human (pro)renin receptor in Syn-hPRR mice.

Abstract P146: Serum Soluble (Pro)Renin Receptor Levels in Patients Undergoing Hemodialysis

Background: The (pro)renin receptor [(P)RR] plays an important role in regulating the tissue renin-angiotensin system (RAS) through the non-proteolytic activation of prorenin, the precursor of renin. (P)RR is cleaved by furin to generate soluble (P)RR [s(P)RR], which is secreted into the extracellular space, and serum s(P)RR has been reported to reflect the status of tissue RAS. Hemodialysis (HD) patients have poor prognosis due to increased prevalence of cardiovascular diseases resulting from severe atherosclerosis. It is speculated that activation of tissue RAS by (P)RR may be associated with this condition, it remains speculative. In the present study, we investigated the relationships between serum s(P)RR levels and background factors including indices of atherosclerosis in HD patients. Methods: Serum s(P)RR levels were measured in 258 maintenance HD patients and these values were compared with 25 subjects with normal renal function. Western blot analysis was done using HD waste water samples, and clearance of s(P)RR through the membrane of dialyzer was examined. Furthermore, relationships between serum s(P)RR levels and background factors were assessed in HD patients. Results: Serum s(P)RR levels were significantly higher in HD patients (30.4±6.1 ng/ml) than those in subjects with normal renal function (16.5±4.3, P<0.0001). s(P)RR was detected in HD waste water by Western blot analysis, and the clearance of s(P)RR and creatinine were 56.9±33.5 and 147.6±9.50 ml/min, respectively. Serum (P)RR levels were significantly higher in those with ankle-brachial index (ABI) of < 0.9, an indicator of severe stenosis or obstruction of lower limb arteries, than those of ≧0.9 (32.2±5.9 and 30.1±6.2 ng/ml, respectively; P<0.05). The association between low ABI and high serum s(P)RR levels were observed even after the correction with age, history of smoking, HbA1c, and LDL-C. Conclusions: Serum s(P)RR levels are significantly higher in HD patients when compared with subjects with normal renal function, although s(P)RR are dialyzed to some extent. High serum s(P)RR levels may be associated with atherosclerosis independent of other risk factors, suggesting that serum s(P)RR could be used as a marker for atherosclerotic condition in HD patients.

Maternal insulin-like growth factor 1 and 2 differentially affect the renin–angiotensin system during pregnancy in the guinea pig

ObjectiveInsulin-like growth factors (IGFs) are known to interact with the renin–angiotensin system (RAS). We previously demonstrated that administration of IGF1 to guinea pigs in early to mid pregnancy promotes placental function and fetal growth in mid to late gestation. Early administration of IGF2 had sustained, but not acute, effects on these parameters and also on placental structural differentiation. Here, we aimed to determine whether the IGFs interact with the placental RAS in early to mid gestation to modulate placental development and increase fetal growth and survival, and if IGF2 binding the IGF2R is implicated in the sustained effects of IGF2 treatment. DesignAt day 20 of pregnancy, guinea pigs were infused with 1mg/kg/day of IGF1, IGF2, Leu27IGF2 or vehicle for 18days and sacrificed on either day 62 (late pregnancy) or during the infusion period on day 35 (early–mid pregnancy). Placental structure at day 35 was analyzed using morphometric technique and expression of RAS genes in the placenta and placental and plasma renin activity were measured at both time points. ResultsCompared with vehicle at day 35 of gestation, IGF1 infusion reduced the total midsagittal cross-sectional area of the placenta (−17%, p=0.02) and the labyrinth area (−22%, p=0.014) but did not alter the labyrinth volume nor labyrinth:interlobium ratios. IGF2 treatment did not affect placental structure.IGF1 did not alter placental mRNA for any of the RAS genes quantified at day 35 (AGTR1, ACE, AGT, TGFB1) but increased TGFB1 expression by more than 16-fold (p=0.005) at day 62. IGF2 increased placental expression of AGTR1 (+88%, p=0.03) and decreased AGT (−73%, p=0.01) compared with the vehicle-treated group at day 35, and both IGF2 and Leu27IGF2 increased expression of TGFB1 at day 62 by 9-fold (p=0.016) and 6-fold (p=0.019) respectively.Both IGFs increased the ratio of active:total placental renin protein (+22% p=0.026 p=0.038) compared to vehicle compared to vehicle at day 35 but not 62. At day 62, IGF2-treated mothers showed a marked increase in total plasma renin (+495%) and active renin (+359%) compared to vehicle but decreased the ratio of active to total renin by 41% (p=0.042). Leu27IGF2-treated animals had higher levels of placental active renin (+73%, p=0.001) and total renin (+71%, p=0.001) compared with the vehicle control. ConclusionsThe data obtained in the current study suggest the potential for alternate roles for the induction of the RAS after IGF treatment. IGF1 and 2 treatments increase the activation of prorenin to renin in the placenta, possibly due to increased protease activity. In addition, IGF2 treatment in early pregnancy may enhance the maternal adaptation to pregnancy through stimulation of renin in the kidney. The sustained effects on placental differentiation and function after IGF2 treatment suggest therapeutic potential for exogenous administration of IGFs in improving pregnancy outcomes.

Regulation of nonclassical renin-angiotensin system receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.

The involvement of the nonclassical renin-angiotensin system (RAS) in the adrenomedullary response to stress is unclear. Therefore, we examined basal and immobilization stress (IMO)-triggered changes in gene expression of the classical and nonclassical RAS receptors in the rat adrenal medulla, specifically the angiotensin II type 2 (AT2) and type 4 (AT4) receptors, (pro)renin receptor [(P)RR], and Mas receptor (MasR). All RAS receptors were identified, with AT2 receptor mRNA levels being the most abundant, followed by the (P)RR, AT1A receptor, AT4 receptor, and MasR. Following a single IMO, AT2 and AT4 receptor mRNA levels decreased by 90 and 50%, respectively. Their mRNA levels were also transiently decreased by repeated IMO. MasR mRNA levels displayed a 75% transient decrease as well. Conversely, (P)RR mRNA levels were increased by 50% following single or repeated IMO. Because of its abundance, the function of the (P)RR was explored in PC-12 cells. Prorenin activation of the (P)RR increased phosphorylation of extracellular signal-regulated kinase 1/2 and tyrosine hydroxylase at Ser(31), likely increasing its enzymatic activity and catecholamine biosynthesis. Together, the broad and dynamic changes in gene expression of the nonclassical RAS receptors implicate their role in the intricate response of the adrenomedullary catecholaminergic system to stress.