RESUMEN
Information on the regional concentrations of angiotensin (Ang) II and its type-1 and -2 receptors (AT(1)R, AT(2)R) in the kidney is still incomplete. Published data on the levels of arterially delivered Ang I and II (Ang Ia, Ang IIa) and intrarenally produced Ang I and II (Ang Ii, Ang IIi) in the renal vein and in whole tissue were analyzed by using a kinetic model of Ang production and distribution in the glomerular and peritubular cortical tissue regions (Glom, Pt). (1) 90% of Ang II is cell-associated, due to its binding to AT(1)R and AT(2)R; (2) most Ang II in the renal cortex is Ang IIi; (3) Ang IIa is mainly localized in Glom; (4) Ang Ii rather than Ang Ia is a substrate of renal angiotensin-converting enzyme; (5) Ang IIi is localized in Pt and its concentration in interstitial fluid is 5-15 times the Ang II concentration in arterial plasma; and (6) in Glom the interstitial concentration of cell surface-bound AT(1)R is above 200K(d), and in Pt the AT(1)R and AT(2)R concentrations are above 10K(d). In conclusion, endocrine Ang II mainly acts in Glom, whereas Pt is exposed to paracrine Ang II generated by the conversion of intrarenally produced Ang I. High AT(1)R concentrations in Glom and Pt favor diffusion-limited binding, so that the apparent binding rate constant at sites closest to the source of Ang II delivery is greatly increased. Results may explain why the kidney is responsive to low levels of endocrine Ang II, despite its high content of paracrine Ang II.
Asunto(s)
Angiotensina II/metabolismo , Riñón/metabolismo , Modelos Biológicos , Circulación Renal , Sistema Renina-Angiotensina , Angiotensina I/análisis , Angiotensina I/biosíntesis , Angiotensina I/metabolismo , Angiotensina II/análisis , Angiotensina II/biosíntesis , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Vasos Sanguíneos/fisiología , Riñón/irrigación sanguínea , Riñón/química , Cinética , Peptidil-Dipeptidasa A/efectos de los fármacos , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/análisis , Receptor de Angiotensina Tipo 2/metabolismo , PorcinosRESUMEN
Information on the levels of angiotensin II (Ang II) and its receptors in the various renal tissue compartments is still incomplete. A model is presented describing the kinetics of Ang II production, distribution, and disposal in the renal cortex. Basic features are: (1) the model is designed to derive, from Ang II measurements in blood and in whole tissue, estimates of the local densities of the Ang II type 1 (AT(1)) and type 2 (AT(2)) receptors, and to calculate the concentrations of endocrine and paracrine Ang II they actually 'see'; (2) glomerular and peritubular tissue are conceived as separate regions (glomerular region (Glom), peritubular region (Pt)); (3) in Glom and in Pt, Ang II is homogeneously distributed in capillary blood and in interstitial fluid; (4) the model allows for local Ang II concentration gradients between interstitium and blood; (5) Ang II from the circulation diffuses into the interstitium of Glom after convective transcapillary transport; (6) Ang II produced in tubules or Pt enters the microcirculation through diffusive overflow from interstitium; (7) the presence of cell-surface-bound Ang II depends on the reaction with AT(1) and AT(2) receptors, and the presence of intracellular Ang II depends on the internalization of Ang II - AT(1) receptor complex; and (8) the model provides for glomerular filtration, vasopeptidase-mediated degradation, and intracellular degradation as mechanisms of elimination. This model can serve as a framework for detailed quantitative studies of the renin-angiotensin system in the kidney.
Asunto(s)
Angiotensina II/metabolismo , Riñón/metabolismo , Modelos Biológicos , Circulación Renal , Sistema Renina-Angiotensina , Angiotensina II/análisis , Angiotensina II/biosíntesis , Animales , Humanos , Riñón/irrigación sanguínea , Riñón/química , Cinética , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismoRESUMEN
BACKGROUND: Both local production and angiotensin II subtype 1 (AT1) receptor-mediated uptake from the circulation contribute to the high levels of angiotensin (Ang) II in the kidney. It is largely unknown where Ang II is produced in the kidney and how much of it originates from the circulation. METHODS: The concentrations of endogenous and 125I-labeled Ang I and II were measured in renal tissue and in blood from pigs receiving systemic infusions of 125I-Ang I. Pigs were either untreated or treated with the angiotensin converting enzyme (ACE) inhibitor captopril or the AT1 receptor antagonist eprosartan. RESULTS: 125I-Ang I was undetectable in renal tissue but the steady-state concentrations of 125I-Ang II in cortical and medullary tissue were four and two times the concentration in arterial blood plasma, respectively. The tissue concentrations of endogenous Ang II were 100 and 60 times higher than in arterial plasma. Eprosartan reduced 125I-Ang II accumulation by 90%, but did not lower tissue Ang II. Captopril did not alter either 125I-Ang II accumulation or tissue Ang II. CONCLUSIONS: The bulk of Ang II in the kidney is cell-associated. The high tissue/blood concentration ratio of endogenous Ang II may depend on the same mechanism as demonstrated for 125I-Ang II, that is, AT1 receptor-mediated binding to cells and endocytosis. If so, the results indicate that most renal AT1 receptors are exposed to locally generated Ang II rather than Ang II from the circulation. We propose the existence of a low-Ang II vascular system-related interstitial compartment that is separate from tubular fluid, where, according to micropuncture studies, Ang II levels might be high.
Asunto(s)
Angiotensina II/metabolismo , Riñón/metabolismo , Tiofenos , Acrilatos/farmacología , Angiotensina II/antagonistas & inhibidores , Angiotensina II/biosíntesis , Angiotensina II/sangre , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Captopril/farmacología , Imidazoles/farmacología , Riñón/citología , Porcinos , Distribución TisularRESUMEN
BACKGROUND: Renal artery stenosis is among the most common curable causes of hypertension. The definitive diagnosis is made by renal angiography, an invasive and costly procedure. The prevalence of renal artery stenosis is less than 1% in non-selected hypertensive patients but is higher when hypertension is resistant to drugs. OBJECTIVE: To study the usefulness of standardised two-drug regimens for identifying drug-resistant hypertension as a predictor of renal artery stenosis. DESIGN AND SETTING: Prospective cohort study carried out in 26 hospitals in The Netherlands. PATIENTS: Patients had been referred for analysis of possible secondary hypertension or because hypertension was difficult to treat. Patients < or =40 years of age were assigned to either amlodipine 10 mg or enalapril 20 mg, and patients >40 years to either amlodipine 10 mg combined with atenolol 50 mg or to enalapril 20 mg combined with hydrochlorothiazide 25 mg. Renal angiography was performed: (1) if hypertension was drug-resistant, ie if diastolic pressure remained > or =95 mm Hg at three visits 1-3 weeks apart or an extra drug was required, and/or (2) if serum creatinine rose by > or =20 micromol/L (> or =0.23 mg/dL) during ACE inhibitor treatment. RESULTS: Of the 1106 patients with complete follow-up, 1022 had been assigned to either the amlodipine- or enalapril-based regimens, 772 by randomisation. Drug-resistant hypertension, as defined above, was identified in 41% of the patients, and 20% of these had renal artery stenosis. Renal function impairment was observed in 8% of the patients on ACE inhibitor, and this was associated with a 46% prevalence of renal artery stenosis. In the randomised patients, the prevalence of renal artery stenosis did not differ between the amlodipine- and enalapril-based regimens. CONCLUSIONS: In the diagnostic work-up for renovascular hypertension the use of standardised medication regimens of maximally two drugs, to identify patients with drug-resistant hypertension, is a rational first step to increase the a priori chance of renal artery stenosis. Amlodipine- or enalapril-based regimens are equally effective for this purpose.
Asunto(s)
Antihipertensivos/uso terapéutico , Hipertensión Renal/tratamiento farmacológico , Hipertensión Renal/etiología , Obstrucción de la Arteria Renal/complicaciones , Obstrucción de la Arteria Renal/diagnóstico por imagen , Adolescente , Adulto , Anciano , Amlodipino/uso terapéutico , Atenolol/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Estudios de Cohortes , Resistencia a Medicamentos , Quimioterapia Combinada , Enalapril/uso terapéutico , Femenino , Humanos , Hidroclorotiazida/uso terapéutico , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Radiografía , Arteria Renal/diagnóstico por imagenRESUMEN
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.
Asunto(s)
Endotelio Vascular/metabolismo , Precursores Enzimáticos/metabolismo , Receptor IGF Tipo 2/fisiología , Renina/metabolismo , Angiotensinas/biosíntesis , Células Cultivadas , Frío , Endocitosis , Precursores Enzimáticos/genética , Humanos , Cinética , Mutación , Renina/genéticaRESUMEN
OBJECTIVES: Recent studies in homogenized hearts suggest that chymase rather than angiotensin converting enzyme (ACE) is responsible for cardiac angiotensin I to angiotensin II conversion. We investigated in intact rat hearts whether (i) enzymes other than ACE contribute to angiotensin I to angiotensin II conversion and (ii) the localization (endothelial/extra-endothelial) of converting enzymes. DESIGN AND METHODS: We used a modified version of the rat Langendorff heart, allowing separate collection of coronary effluent and interstitial fluid. Hearts were perfused with angiotensin I (arterial concentration 5-10 pmol/ml) under control conditions, in the presence of captopril (1 micromol/l) or after endothelium removal with 0.2% triton X-100. Endothelium removal was verified as the absence of a coronary vasodilator response to 10 nmol bradykinin. Angiotensin I and angiotensin II were measured in coronary effluent and interstitial fluid with sensitive radioimmunoassays. RESULTS: In control hearts, 45% of arterial angiotensin I was metabolized during coronary passage, partly through conversion to angiotensin II. At steady-state, the angiotensin I concentration in interstitial fluid was three to four-fold lower than in coronary effluent, while the angiotensin II concentrations in both fluids were similar. Captopril and endothelium removal did not affect coronary angiotensin I extraction, but increased the interstitial fluid levels of angiotensin I two- and three-fold, respectively, thereby demonstrating that metabolism (by ACE) as well as the physical presence of the endothelium normally prevent arterial angiotensin I from reaching similar levels in coronary effluent and interstitial fluid. Captopril, but not endothelium removal, greatly reduced the angiotensin II levels in coronary effluent and interstitial fluid. With the ACE inhibitor, the angiotensin II/I ratios in coronary effluent and interstitial fluid were 83 and 93% lower, while after endothelium removal, the ratios were 33 and 71% lower. CONCLUSIONS: In the intact rat heart, ACE is the main contributor to angiotensin I to angiotensin II conversion, both in the coronary vascular bed and the interstitium. Cardiac ACE is not limited to the coronary vascular endothelium.
Asunto(s)
Angiotensina II/metabolismo , Angiotensina I/metabolismo , Miocardio/metabolismo , Peptidil-Dipeptidasa A/fisiología , Angiotensina I/sangre , Angiotensina I/farmacología , Angiotensina II/sangre , Animales , Circulación Coronaria/fisiología , Vasos Coronarios/metabolismo , Endotelio Vascular/fisiología , Espacio Extracelular/metabolismo , Masculino , Perfusión , Ratas , Ratas Wistar , Distribución TisularRESUMEN
OBJECTIVES: To investigate whether tissue angiotensin II generation occurs intra- or extracellularly, we studied the subcellular localization of angiotensin II in kidney and adrenal, two organs with high endogenous angiotensin II concentrations. DESIGN AND METHODS: Tissues were obtained, following a 1 h infusion of 125I-angiotensin I or 125I-angiotensin II to simultaneously determine the localization of plasma-derived angiotensin II, from five control pigs and four pigs that had been pretreated with the AT1 receptor antagonist eprosartan. Subcellular organelles, prepared by differential centrifugation from homogenized tissue, were characterized using organelle-specific markers. RESULTS: 125I-angiotensin II and angiotensin II were present in all organelles, with identical distribution profiles. In mitochondria-enriched fractions the relative specific activities [RSAs = (concentration per mg protein in fraction)/(concentration per mg protein in homogenate)] of the two peptides were similar to those in homogenate, whereas in cytosol-enriched fractions their RSAs were five- to 10-fold lower (P< 0.05 versus homogenate). In microsome- as well as in lysosome-enriched fractions the RSAs of 125I-angiotensin II and angiotensin II were two- to four-fold higher than in homogenate (P < 0.05), and their RSAs were also higher in renal nuclei-enriched fractions (P< 0.05). Eprosartan increased plasma angiotensin II to a larger degree than tissue angiotensin II and greatly reduced tissue 125I-angiotensin II. This led to similar decreases in the tissue/plasma concentration ratios of 125I-angiotensin II and angiotensin II. The subcellular distribution of both angiotensin II peptides was not affected by eprosartan. CONCLUSIONS: Local angiotensin II synthesis in adrenal and kidney occurs predominantly extracellularly, and is followed by rapid AT1 receptor-mediated endocytosis, thereby leading to high intracellular angiotensin II levels.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Angiotensina II/metabolismo , Riñón/metabolismo , Fracciones Subcelulares/metabolismo , Tiofenos , Acrilatos/farmacología , Angiotensina II/antagonistas & inhibidores , Angiotensina II/sangre , Antagonistas de Receptores de Angiotensina , Animales , Femenino , Imidazoles/farmacología , Masculino , Porcinos , Distribución TisularRESUMEN
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.
Asunto(s)
Precursores Enzimáticos/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Receptor IGF Tipo 2/metabolismo , Renina/metabolismo , Líquido Amniótico/enzimología , Animales , Animales Recién Nacidos , Células Cultivadas , Activación Enzimática , Fibroblastos/metabolismo , Humanos , Riñón/enzimología , Cinética , Inhibidores de Proteasas/farmacología , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Precursores Enzimáticos/metabolismo , Miocardio/metabolismo , Receptor IGF Tipo 2/metabolismo , Renina/metabolismo , Líquido Amniótico , Angiotensina II/biosíntesis , Animales , Animales Recién Nacidos , Sangre , Células Cultivadas , Activación Enzimática , Femenino , Líquido Folicular , Glicosilación , Humanos , Hipertensión/sangre , Hipertensión/metabolismo , Riñón/metabolismo , Masculino , Ovario/metabolismo , Fosforilación , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Obstrucción de la Arteria Renal/sangre , Obstrucción de la Arteria Renal/metabolismoRESUMEN
BACKGROUND: The mechanisms behind the beneficial effects of renin-angiotensin system blockade after myocardial infarction (MI) are not fully elucidated but may include interference with tissue angiotensin II (Ang II). METHODS AND RESULTS: Forty-nine pigs underwent coronary artery ligation or sham operation and were studied up to 6 weeks. To determine coronary angiotensin I (Ang I) to Ang II conversion and to distinguish plasma-derived Ang II from locally synthesized Ang II, (125)I-labeled and endogenous Ang I and II were measured in plasma and in infarcted and noninfarcted left ventricle (LV) during (125)I-Ang I infusion. Ang II type 1 (AT(1)) receptor-mediated uptake of circulating (125)I-Ang II was increased at 1 and 3 weeks in noninfarcted LV, and this uptake was the main cause of the transient elevation in Ang II levels in the noninfarcted LV at 1 week. Ang II levels and AT(1) receptor-mediated uptake of circulating Ang II were reduced in the infarct area at all time points. Coronary Ang I to Ang II conversion was unaffected by MI. Captopril and the AT(1) receptor antagonist eprosartan attenuated postinfarct remodeling, although both drugs increased cardiac Ang II production. Captopril blocked coronary conversion by >80% and normalized Ang II uptake in the noninfarcted LV. Eprosartan did not affect coronary conversion and blocked cardiac Ang II uptake by >90%. CONCLUSIONS: Both circulating and locally generated Ang II contribute to remodeling after MI. The rise in tissue Ang II production during angiotensin-converting enzyme inhibition and AT(1) receptor blockade suggests that the antihypertrophic effects of these drugs result not only from diminished AT(1) receptor stimulation but also from increased stimulation of growth-inhibitory Ang II type 2 receptors.
Asunto(s)
Modelos Cardiovasculares , Infarto del Miocardio/sangre , Peptidil-Dipeptidasa A/sangre , Receptores de Angiotensina/sangre , Angiotensina II/sangre , Animales , Captopril/sangre , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , PorcinosRESUMEN
BACKGROUND: Patients with hypertension and renal-artery stenosis are often treated with percutaneous transluminal renal angioplasty. However, the long-term effects of this procedure on blood pressure are not well understood. METHODS: We randomly assigned 106 patients with hypertension who had atherosclerotic renal-artery stenosis (defined as a decrease in luminal diameter of 50 percent or more) and a serum creatinine concentration of 2.3 mg per deciliter (200 micromol per liter) or less to undergo percutaneous transluminal renal angioplasty or to receive drug therapy. To be included, patients also had to have a diastolic blood pressure of 95 mm Hg or higher despite treatment with two antihypertensive drugs or an increase of at least 0.2 mg per deciliter (20 micromol per liter) in the serum creatinine concentration during treatment with an angiotensin-converting-enzyme inhibitor. Blood pressure, doses of antihypertensive drugs, and renal function were assessed at 3 and 12 months, and patency of the renal artery was assessed at 12 months. RESULTS: At base line, the mean (+/-SD) systolic and diastolic blood pressures were 179+/-25 and 104+/-10 mm Hg, respectively, in the angioplasty group and 180+/-23 and 103+/-8 mm Hg, respectively, in the drug-therapy group. At three months, the blood pressures were similar in the two groups (169+/-28 and 99+/-12 mm Hg, respectively, in the 56 patients in the angioplasty group and 176+/-31 and 101+/-14 mm Hg, respectively, in the 50 patients in the drug-therapy group; P=0.25 for the comparison of systolic pressure and P=0.36 for the comparison of diastolic pressure between the two groups); at the time, patients in the angioplasty group were taking 2.1+/-1.3 defined daily doses of medication and those in the drug-therapy group were taking 3.2+/-1.5 daily doses (P<0.001). In the drug-therapy group, 22 patients underwent balloon angioplasty after three months because of persistent hypertension despite treatment with three or more drugs or because of a deterioration in renal function. According to intention-to-treat analysis, at 12 months, there were no significant differences between the angioplasty and drug-therapy groups in systolic and diastolic blood pressures, daily drug doses, or renal function. CONCLUSIONS: In the treatment of patients with hypertension and renal-artery stenosis, angioplasty has little advantage over antihypertensive-drug therapy.
Asunto(s)
Angioplastia de Balón , Antihipertensivos/uso terapéutico , Arteriosclerosis/terapia , Hipertensión Renovascular/terapia , Obstrucción de la Arteria Renal/terapia , Anciano , Presión Sanguínea/efectos de los fármacos , Creatinina/sangre , Femenino , Humanos , Hipertensión Renovascular/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Radiografía , Arteria Renal/diagnóstico por imagen , Obstrucción de la Arteria Renal/diagnóstico por imagenRESUMEN
To assess the importance for vasoconstriction of in situ angiotensin (Ang) II generation, as opposed to Ang II delivery via the circulation, we determined forearm vasoconstriction in response to Ang I (0.1 to 10 ng. kg(-1). min(-1)) and Ang II (0.1 to 5 ng. kg(-1). min(-1)) in 14 normotensive male volunteers (age 18 to 67 years). Changes in forearm blood flow (FBF) were registered with venous occlusion plethysmography. Arterial and venous blood samples were collected under steady-state conditions to quantify forearm fractional Ang I-to-II conversion. Ang I and II exerted the same maximal effect (mean+/-SEM 71+/-4% and 75+/-4% decrease in FBF, respectively), with similar potencies (mean EC(50) [range] 5.6 [0.30 to 12.0] nmol/L for Ang I and 3.6 [0.37 to 7.1] nmol/L for Ang II). Forearm fractional Ang I-to-II conversion was 36% (range 18% to 57%). The angiotensin-converting enzyme (ACE) inhibitor enalaprilat (80 ng. kg(-1). min(-1)) inhibited the contractile effects of Ang I and reduced fractional conversion to 1% (0.1% to 8%), thereby excluding a role for Ang I-to-II converting enzymes other than ACE (eg, chymase). The Ang II type 1 receptor antagonist losartan (3 mg. kg(-1). min(-1)) inhibited the vasoconstrictor effects of Ang II. In conclusion, the similar potencies of Ang I and II in the forearm, combined with the fact that only one third of arterially delivered Ang I is converted to Ang II, suggest that in situ-generated Ang II is more important for vasoconstriction than circulating Ang II. Local Ang II generation in the forearm depends on ACE exclusively and results in vasoconstriction via Ang II type 1 receptors.
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Angiotensina II/administración & dosificación , Angiotensina II/biosíntesis , Angiotensina I/administración & dosificación , Endotelio Vascular/enzimología , Antebrazo/irrigación sanguínea , Peptidil-Dipeptidasa A/metabolismo , Adolescente , Adulto , Anciano , Angiotensina I/sangre , Angiotensina II/sangre , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Antihipertensivos/administración & dosificación , Arteria Braquial/fisiología , Enalaprilato/administración & dosificación , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Humanos , Losartán/administración & dosificación , Masculino , Persona de Mediana Edad , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/metabolismo , Flujo Sanguíneo Regional , Vasoconstricción/fisiologíaRESUMEN
OBJECTIVE: The hypertrophic response of cardiomyocytes exposed to mechanical stretch is assumed to depend on the release of angiotensin (Ang) II from these cells. Here we studied the synthesis of renin-angiotensin system (RAS) components by cardiac cells under basal conditions and after stretch. METHODS: Myocytes and fibroblasts were isolated by enzymatic dissociation from hearts of 1-3-day-old Wistar rat strain pups, grown for 1 day in serum-supplemented medium and then cultured in a chemically defined, serum-free medium. Medium and cell lysate were collected 5 days later or after exposure of the cells to cyclic stretch for 24 h. Prorenin, renin and angiotensinogen were measured by enzyme-kinetic assay; Ang I and Ang II were measured by radioimmunoassay after SepPak extraction and HPLC separation. RESULTS: Prorenin, but none of the other RAS components, could be detected in the medium of both cell types. However, its levels were low and the Ang I-generating activity corresponding with these low prorenin levels could not be inhibited by the specific rat renin inhibitor CH-732, suggesting that it was most likely due to bovine and/or horse prorenin sequestered from the serum-containing medium to which the cells had been exposed prior to the serum-free period. When incubated with Ang I, both myocytes and fibroblasts generated Ang II in a captopril-inhibitable manner. Myocyte and fibroblast cell lysates did not contain prorenin, renin, angiotensinogen, Ang I or Ang II in detectable quantities. Stretch increased myocyte protein synthesis by 20%, but was not accompanied by Ang II release into the medium. CONCLUSION: Cardiac myocytes and fibroblasts do not synthesize renin, prorenin or angiotensinogen in concentrations that are detectable or, it not detectable, high enough to result in Ang II concentrations of physiological relevance. These cells do synthesize ACE, thereby allowing the synthesis of Ang II at cardiac tissue sites when renin and angiotensinogen are provided via the circulation. Ang II is not a prerequisite to observe a hypertrophic response of cardiomyocytes following stretch.
Asunto(s)
Angiotensina II/biosíntesis , Miocardio/metabolismo , Miocardio/patología , Peptidil-Dipeptidasa A/metabolismo , Análisis de Varianza , Angiotensina I/análisis , Angiotensina II/análisis , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Angiotensinógeno/análisis , Animales , Animales Recién Nacidos , Captopril/farmacología , Células Cultivadas , Medio de Cultivo Libre de Suero , Precursores Enzimáticos/análisis , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Hipertrofia , Ratas , Ratas Wistar , Renina/análisis , Estrés MecánicoRESUMEN
AIMS/HYPOTHESIS: The renin-angiotensin system is possibly involved in the pathogenesis of diabetic nephropathy. The most striking change in renin-angiotensin system components in blood of patients with diabetic nephropathy is an increased prorenin concentration. We investigated prospectively serum concentrations of renin-angiotensin system components and the time course of prorenin increase in normoalbuminuric diabetic patients developing microalbuminuria. METHODS: Patients (n = 199) with Type I (insulin-dependent) diabetes mellitus and normoalbuminuria at baseline were prospectively followed for 10 years. The prorenin concentrations and other variables possibly associated with the occurrence of microalbuminuria, were investigated by Cox-regression analysis. RESULTS: Of the patients 29 developed microalbuminuria. Glycated haemoglobin values were higher at baseline in these patients. Serum prorenin was similar at baseline but rose in the 29 patients before the development of microalbuminuria and was stable in patients with stable albumin excretion. Renin, angiotensinogen and angiotensin converting enzyme serum concentrations were stable in both groups. Prorenin and glycated haemoglobin were independent prognostic factors for the development of microalbuminuria. A prognostic index, based on these variables, was constructed to estimate the relative risk of developing microalbuminuria. CONCLUSIONS/INTERPRETATION: Increase in serum prorenin precedes onset of microalbuminuria in normotensive patients with insulin-dependent diabetes mellitus. High concentrations of prorenin in combination with high values of glycated haemoglobin can be used as a predictor of development of microalbuminuria.
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Albuminuria/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Precursores Enzimáticos/sangre , Renina/sangre , Adulto , Angiotensinógeno/sangre , Biomarcadores/sangre , Presión Sanguínea , Estudios de Cohortes , Dinamarca , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/orina , Femenino , Estudios de Seguimiento , Hemoglobina Glucada/análisis , Humanos , Masculino , Peptidil-Dipeptidasa A/sangre , Valor Predictivo de las Pruebas , Estudios Prospectivos , Análisis de Regresión , Sistema Renina-Angiotensina , Factores de TiempoRESUMEN
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.
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Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/genética , Precursores Enzimáticos/sangre , Variación Genética/fisiología , Renina/sangre , Renina/genética , Adulto , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Retinopatía Diabética/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético/fisiologíaRESUMEN
OBJECTIVE: To investigate the mechanisms of vascular uptake of prorenin and renin and to explore the possibility of vascular activation of prorenin. DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Our findings provide evidence for M-6-P receptor-dependent endocytosis of (pro)renin and proteolytic prorenin activation by vascular endothelial cells.
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Endotelio Vascular/metabolismo , Precursores Enzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Renina/metabolismo , Análisis de Varianza , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Precursores Enzimáticos/inmunología , Humanos , Inmunoensayo , Renina/inmunologíaRESUMEN
BACKGROUND: Our renin IRMA overestimated renin in plasmas with high prorenin-to-renin ratios. We suspected that the overestimation of renin was caused less by cross-reactivity of the renin-specific antibody with prorenin than by a conformational change of prorenin into an enzymatically active form during the assay. METHODS: Because the inactive form of prorenin converts slowly into an active form at low temperature, we raised the assay temperature from 22 degrees C to 37 degrees C, simultaneously shortening the incubation time from 24 to 6 h. The former IRMA was performed in <1 working day with these modifications. RESULTS: The comeasurement of prorenin as renin was eliminated. Reagents were stable at 37 degrees C, and the new and old IRMAs were comparable in terms of precision and accuracy. The functional lower limit of the assay (4 mU/L) was below the lower reference limit (9 mU/L). The modified IRMA agreed closely with the activities measured with an enzyme-kinetic assay. Results were not influenced by the plasma concentration of angiotensinogen. At normal angiotensinogen concentrations, the IRMA closely correlated with the classical enzyme-kinetic assay of plasma renin activity. CONCLUSION: The modified IRMA, performed at 37 degrees C, avoids interference by prorenin while retaining the desirable analytical characteristics of the older IRMA and requiring less time.
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Renina/sangre , Reacciones Cruzadas , Activación Enzimática , Precursores Enzimáticos/sangre , Femenino , Humanos , Masculino , Embarazo , Radioinmunoensayo , Renina/metabolismo , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
OBJECTIVE: To assess inter-observer agreement in the interpretation of renal angiograms. DESIGN: Comparison of the assessment of renal angiograms by three experienced radiologists, who evaluated the number of renal arteries and the presence, location, aspect and severity of a renal artery stenosis. SETTING: General hospital and university hospital serving urban and rural populations. PATIENTS: Patients with difficult-to-treat hypertension referred for diagnostic work-up; 312 angiograms with the intra-arterial digital subtraction technique were obtained from 289 consecutive patients. MAIN OUTCOME MEASURES: Inter-observer agreement was tested for the following parameters: number of arteries per kidney, presence of stenosis, location of stenosis (truncal, ostial), aspect of stenosis (concentric, eccentric, post-stenotic dilatation), severity of stenosis (reduction of lumen diameter in categories of 30%, 40%, etc. to 100%), and overall quality of the angiographic images. Kappa (kappa) values and weighted kappa between the three pairs of radiologists were used as estimates of inter-observer agreement RESULTS: Agreement about the number of renal arteries was reasonable (kappa = 0.50-0.72), as was agreement about the presence of stenosis (kappa = 0.68-0.86). Agreement about stenosis location and aspect was poor (kappa = 0.26-0.47 and kappa = 0.15-0.26, respectively). There was general agreement about the severity of stenosis (weighted kappa = 0.65-0.70), but it was not possible to distinguish between 50 and 60% stenosis or between 60 and 70% stenosis (kappa < 0.40). No correlation was found between agreement on severity of stenosis and the quality of the images. CONCLUSIONS: It is not realistic to make statements about what degree of renal artery stenosis is clinically significant, as long as the intra-arterial angiogram with digital subtraction remains the gold standard. It is likewise risky to rely too strongly on stenosis morphology as visualized by renal angiography in choosing between balloon angioplasty and stent deployment.
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Angiografía/estadística & datos numéricos , Hipertensión Renal/epidemiología , Obstrucción de la Arteria Renal/epidemiología , Adulto , Humanos , Hipertensión Renal/diagnóstico por imagen , Persona de Mediana Edad , Variaciones Dependientes del Observador , Radiología/estadística & datos numéricos , Obstrucción de la Arteria Renal/diagnóstico por imagen , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To assess the importance, for vasoconstriction, of in situ angiotensin (Ang) II generation, as opposed to ang II delivery to AT receptors via the organ bath fluid. METHODS: Ang I and II concentration-response curves in human and porcine coronary arteries (HCAs, PCAs) were constructed in relation to estimates of the clearances of Ang I and II (ClAngI, ClAngII) from the organ bath and the release of newly formed Ang II (RAngII) into the bath fluid. HCAs were from 25 heart valve donors (age 5-54 years), and PCAs from 14 pigs (age 3 months). RESULTS: Ang I- and II-evoked constrictions were inhibited by the AT1 receptor antagonist, irbesartan, and were not influenced by the AT2 receptor antagonist, PD123319. In HCAs Ang II was only three times more potent than Ang I, wheres, in the experiments with Ang I, comparison of ClAngI with ClAngII and RAngII indicated that most of the arterially produced Ang II did not reach the bath fluid. Also in PCAs Ang I and II showed similar potency. In HCAs both the ACE inhibitor, captopril, and the chymase inhibitor, chymostatin, inhibited Ang I-evoked vasoconstriction, while only chymostatin had a significant effect on ClAngI. In PCAs Ang I-evoked vasoconstriction was almost completely ACE-dependent. CONCLUSIONS: This study points towards the functional importance of in situ ACE- and chymase-dependent Ang II generation, as opposed to Ang II delivery via the circulation. It also indicates that functionally relevant changes in local Ang I-II conversion are not necessarily reflected by detectable changes in circulating Ang II.
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Angiotensina II/fisiología , Vasos Coronarios/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Serina Endopeptidasas/metabolismo , Vasoconstricción/efectos de los fármacos , Adolescente , Adulto , Angiotensina I/análisis , Angiotensina II/análisis , Angiotensina II/biosíntesis , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Compuestos de Bifenilo/farmacología , Captopril/farmacología , Niño , Preescolar , Quimasas , Vasos Coronarios/efectos de los fármacos , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Imidazoles/farmacología , Irbesartán , Masculino , Persona de Mediana Edad , Oligopéptidos/farmacología , Piridinas/farmacología , Porcinos , Tetrazoles/farmacologíaRESUMEN
OBJECTIVE: The angiotensin-converting enzyme (ACE) gene I/D polymorphism accounts for part of the variation in ACE concentration; subjects with one or two D alleles have approximately 25 and 50% higher ACE levels, respectively, than subjects with two I alleles. Data from studies on the pressor effects of angiotensin (Ang) I in DD compared with II subjects are inconsistent, because enhanced conversion in DD subjects may have been masked by a decreased responsiveness to Ang II. Here we quantify ACE genotype-related Ang I to Ang II conversion in the human forearm and leg using non-pressor 125I-Ang I infusions. DESIGN AND METHODS: Infusions were given to 12 women and 17 men (age 24-67 years) who were undergoing renal vein sampling followed by renal angiography for diagnostic purposes. 125I-Ang I was infused for 20 min into the right antecubital vein, and blood samples for the measurement of 125I-labelled and endogenous Ang I and Ang II were taken from the aorta, the left antecubital vein and a femoral vein under steady-state conditions. Genotype frequencies were determined by polymerase chain reaction. RESULTS: Fractional conversion (i.e. the percentage of arterially delivered 125I-Ang I that is converted to 125I-Ang II) in the forearm (38+/-4, 30+/-3 and 31+/-6% in 8 II, 16 ID and 5 DD subjects, respectively; mean +/- SEM) and leg (52+/-4, 48+/-3 and 42+/-5%) was similar in all three groups. In addition, no genotype-related differences in plasma Ang II/I ratio (a measure of ACE activity) were observed at the three sampling sites. CONCLUSIONS: Regional Ang I to Ang II conversion does not parallel the previously described D allele-related differences in ACE concentration, suggesting that effects other than enhanced conversion may underlie the reported associations between the D allele and various cardiovascular diseases.