Human prorenin determination by hybrid immunocapture liquid chromatography/mass spectrometry: A mixed-solvent-triggered digestion utilizing D-optimal design.

RATIONALE: Human prorenin, representing the precursor of mature renin, has been discussed as a potential biomarker, e.g. in diagnosing primary hyperaldosteronism or diabetes-induced nephropathy. Currently, only immunoassays are available for prorenin quantification. As the similarity of prorenin to active renin impedes its accurate determination by immunoassay, mass spectrometry appears as an accurate alternative for differentiation of that protein. METHODS: Immunoaffinity purification plus a mixed-solvent-triggered digestion was combined with liquid chromatography/mass spectrometry (LC/MS) to enable a fast, sensitive, and less laboratory-intensive approach to the quantification of prorenin. Statistical experimental planning, which is known as Design of Experiments (DOE), was used to identify the optimal conditions for the generation of the signature peptides within a manageable number of experiments. The efficiency of the mixed-solvent-triggered digestion by trypsin was investigated using four different organic solvents: acetonitrile, acetone, tetrahydrofuran and methanol. RESULTS: By utilizing a D-optimal design, we found that the optimal mixed-solvent type for the generation of both signature peptides was acetonitrile at a concentration of 84% and an incubation temperature of 16°C. Using the mixed-solvent-triggered digestion, the procedure time allowed a fast analysis of active renin and prorenin with a short digestion time of 98 min. This optimized mixed-solvent-triggered digestion procedure was applied to detect renin and prorenin successfully in human plasma by the newly developed hybrid approach. CONCLUSIONS: The identification of unique surrogates for human prorenin enabled the mass spectrometric differentiation between the two similar proteins. The novel hybrid approach successfully proved its ability to purify, detect and distinguish between prorenin and active renin in human plasma.

A quality control system for ligand-binding assay of plasma renin activity: Proof-of-concept within a pharmacodynamic study.

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded "Labeling of Enalapril in Neonates up to Adolescents" project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.

A widely applicable plasma renin activity assay by LC-MS/MS with offline solid phase extraction.

BACKGROUND: The measurement of plasma renin activity using LC-MS/MS is an attractive alternative to radioimmunoassay techniques. A published method for renin activity by LC-MS/MS uses equipment that is not available in all LC-MS/MS laboratories. Here, we present an offline modification to allow users of LC-MS/MS to perform this analysis without specialist equipment. METHODS: Samples were prepared in duplicate and incubated for 6.5 h and 24 h. Solid phase extraction was performed offline using Waters Oasis MAX ion-exchange 96-well plate. A method comparison was performed between this assay and a previously published assay using on-line solid phase extraction. RESULTS: The offline method for plasma rennin activity gave similar results to the on-line method across the concentration range. Analytical performance was also found to be comparable and the use of a 24 h incubation for low samples was also deemed unnecessary. DISCUSSION: The method described is an acceptable alternative to a previously published assay which does not require the use of highly specialist equipment and will be applicable to the majority of LC-MS/MS users.

Detección de renina en carcinomas cromófobos de células renales / Detection of renin in chromophobe renal cell carcinomas

Objetivo: Evaluar la expresión de renina en carcinomas cromófobos de células renales y la posible asociación de esta expresión con hipertensión arterial (HTA) sistémica. Material y métodos: Estudio descriptivo retrospectivo. Se incluyeron todos los casos con diagnóstico confirmado de carcinoma cromófobo entre 1990-2004: 31 casos provenientes de 31 pacientes. De los bloques de tejido tumoral incluidos en parafina se hizo inmunohistoquímica para detectar renina usando un anticuerpo monoclonal. De los archivos de historias clínicas obtuvimos información completa con respecto a la presión arterial sistémica antes y después de la resección tumoral. Comparamos frecuencias de HTA en casos con y sin expresión de renina (prueba de Fisher o χ2, según lo adecuado) y evolución de la HTA posresección. Resultados: En 10 de los 31 tumores (32,3%) hubo inmunotinción para renina; esta tinción fue difusa en 6 casos y focal en los 4 restantes. Se detectó HTA en 6 de los 10 pacientes con expresión de renina (60,0%) y en 6 de los 21 pacientes sin expresión de renina (28,6%) (p=0,13). Después de la resección tumoral, ningún paciente con expresión de renina e HTA mostró remisión de la hipertensión. Conclusión: En el carcinoma cromófobo de células renales es frecuente la expresión de renina, pero esta renina parece clínicamente inactiva. Serán necesarios más estudios para conocer las implicaciones en el diagnóstico, la patogénesis y la presentación clínica de esta expresión (AU)

Aim: To evaluate frequency of renin detection in chromophobe renal cell carcinoma, and if this expression was associated to systemic high blood pressure. Material and methods: A descriptive retrospective study. All the cases with confirmed diagnosis of chromophobe carcinoma and resected between 1990 and 2004 were included in our study: 31 cases from 31 patients. Immunohistochemistry was carried out on sections from the paraffin-embedded tissue using a monoclonal antiserum. Patient blood pressure before and after neoplasm resection was registered from clinical histories. We compared frequencies of hypertension in cases with and without expression of renin (Fisher´s text or χ2 as appropriate) and evolution of HTA after tumour resection. Results: We found that 10 of 31 tumors (32.3%) contained immunoreactivity for renin; this staining was diffuse in 6 cases and focal in the other 4. Systemic hypertension was detected in 6 of 10 (60.0%) patients with renin expression and in 6 of 21 (28.6%) patients without renin immunolabeling (p=0.13). After tumor resection none patient with renin expression and high blood pressure showed remission of the hypertension. Conclusion: Renin is frequently expressed in chromophobe renal cell carcinoma, but this renin appears clinically inactive. More studies will be necessary to know implications of this feature on clinical presentation, diagnosis or pathogenesis (AU)

Cathepsin B is not the processing enzyme for mouse prorenin.

Renin, an aspartyl protease that catalyzes the rate-limiting step in the renin-angiotensin system (RAS), is proteolytically activated by a second protease [referred to as the prorenin processing enzyme (PPE)] before its secretion from the juxtaglomerular cells of the kidney. Although several enzymes are capable of activating renin in vitro, the leading candidate for the PPE in the kidney is cathepsin B (CTSB) due to is colocalization with the renin precursor (prorenin) in juxtaglomerular cell granules and because of its site-selective activation of human prorenin both in vitro and in transfected tissue culture cell models. To verify the role of CTSB in prorenin processing in vivo, we tested the ability of CTSB-deficient (CTSB-/-) mice to generate active renin. CTSB-/- mice do not exhibit any overt symptoms (renal malformation, preweaning mortality) typical of an RAS deficiency and have normal levels of circulating active renin, which, like those in control animals, rise more than 15-fold in response to pharmacologic inhibition of the RAS. The mature renin enzyme detected in kidney lysates of CTSB-/- mice migrates at the same apparent molecular weight as that in control mice, and the processing to active renin is not affected by chloroquine treatment of the animals. Finally, the distribution and morphology of renin-producing cells in the kidney is normal in CTSB-/- mice. In conclusion, CTSB-deficient mice exhibit no differences compared with controls in their ability to generate active renin, and our results do not support CTSB as the PPE in mice.

Purification and characterization of recombinant human renin for X-ray crystallization studies.

BACKGROUND: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies. RESULTS: A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained. CONCLUSION: This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.

Efficient production of recombinant human (pro)renin utilizing a decahistidine tag attached at the C-terminus.

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro.

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg(-1) and 326 U mg(-1) respectively. The optimum pH range was from 7-8.5 at 37 degrees C and the optimum temperature was 50 degrees C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608 +/- 0.07 mM and 249 sec(-1) respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68 +/- 12.55 nM and 6.763 +/- 0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.

A renin-like enzyme in the leech Theromyzon tessulatum.

We report on the biochemical isolation and characterization of a 32 kDa aspartyl protease from the leech Theromyzon tessulatum. Following a three step purification (gel permeation chromatography, pepstatin A-sepharose affinity column separation followed by reversed-phase HPLC) a renin-like enzyme was purified to homogeneity. The first 124 amino acid residues of the N-terminal part of the purified S-pyridylethylated leech renin exhibits a 26.5-35.5% sequence identity with that of mammals. The 20-81 region of leech renin exhibits a 80% sequence homology with the 175-232 region in mammals. This highly conserved region, which is also found in all aspartic proteases, possesses the aspartyl catalytic residue (D11TGSS). Leech renin hydrolyses at neutral pH and at 37 degrees C the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptides, with a specific activity of 115 microg AI/min/mg (K[M] 22 microM; K[cat], 2.7). This hydrolysis is inhibited by pepstatin A (IC50: 4.6 microM). Moreover, this enzyme is found on a multiple hormone precursor of 19 kDa which exhibits a specific activity of 850 pmol AI/min/mg of renin. This is the first biochemical characterization of a renin-like enzyme in invertebrates and non-mammalian vertebrates.

Prorenin activation and prohormone convertases in the mouse As4.1 cell line.

The precise identification of prorenin-processing enzymes has been hampered by the very low abundance of juxtaglomerular cells in the kidney. Recently, an immortalized renin-producing renal tumor cell line (As4.1) has been proposed as a model to carry out such studies. Despite the fact that they contain secretory granules, we found no evidence (on the basis of enzymatic assays of renin activity in the supernatant of the cells and of immunoprecipitations experiments) that the As4.1 cells can secrete active renin through the regulated pathway. As4.1 cells produce only renin-1, as they derive from a strain of mice expressing only one renin gene. However, stable transfection of these cells with a renin-2 expression plasmid increased the capacity of this cell line to secrete active renin in the regulated pathway. Northern blot and reverse transcriptase-polymerase chain reaction amplification (RT-PCR) assays revealed that furin, PACE4 and PC5 were the only members of the proprotein convertase (PC) family to be present in these cells. As PC5 is the only such enzyme with the demonstrated ability to process mouse prorenin 2, it may constitute a candidate enzyme for the processing of prorenin-2 in mouse juxtaglomerular cells. However, it is not likely to be involved in the processing of mouse prorenin 1.

Spontaneous inhibition or inactivation of renin in rat plasma.

Renin appears to be rapidly inactivated in vitro. The present study was undertaken to clarify this observation and to establish the existence of substances involved in renin inactivation. The disappearance rate of renin (including pure renin) was measured in plasma incubated at 37 degrees C and in circulating blood. Pure renin added to plasmas disappears in vitro at the same rate (t1/2 approximately or equal to 40 min) that renin in plasma from normal rats and from rats submitted to a hemorrhage. This process appears not to be mediated by proteases. The disappearance rate of endogenous renin in the normal group (n = 18) was 39.7 min with rapid phase (R) of t1/2 = 14.2 min and a slow phase (S) of t1/2 = 94.3 min), whereas it was 32.1 min (t1/2 R = 13.1 min and t1/2 S = 69.1 min) in rats submitted to an hemorrhage (n = 6). The t1/2 of pure renin was 31.4 min (t1/2 R = 13.3 min and t1/2 S = 69.2 min). Incubation of plasma reveals that renin is inactivated or inhibited in vitro at a similar rate than in circulating plasma. These results suggest that inactivation and sequestration of renin could be two independent mechanisms in the maintenance of plasma renin activity.

Actividad de renina plasmática en enfermedad pulmonar obstructiva crónica

Introducción: En estados avanzados de la EPOC pueden demostrarse alteraciones neurohormonales significativas, que se correlacionan con la aparición de hipercapnia y retención de sodio y agua. No se conoce si hay activación del eje renina-angiotensina-aldosterona en etapas tempranas de la enfermedad, como se ha detectado que ocurre en la enfermedad ventricular izquierda. Objetivo: evaluar la actividad de renina plasmática, un índice de la función del eje renina-angiotensión-aldosterona, en pacientes con EPOC que no han presenta edemas ni descompensación clínica. Pacientes y Métodos: Diez y seis pacientes fueron estudiados con pruebas de función pulmonar (espirometría, curvo flujo-volumen), gasimetría arterial, radiografía del tórax y ecocardiograma transtorácico. En sangre venosa y en condiciones de reposo se determinó actividad de renina plasmática por radioinmunoensayo. Resultados: Se estudiaron 16 pacientes, 14 hombres y 2 mujeres, edad promedio de 61 años con EPOC moderado a severo (promedio del VEF 1 predicho 50 por ciento, relación VEF1/CVF 51 por ciento), PaO2 promedio 52 mm Hg y PaCO2 promedio 34,3 mm Hg, con hipertensión pulmonar moderada a severa (presión sistólica pulmonar promedio 61 mmHg y media pulmonar 41,7) y alteración de la función sistólica del ventriculo derecho. La función ventrícular izquierda presentaba alteración diastólica en 87 por ciento de los sujetos, mientras que la función sistólica era completamente normal, con una fracción de eyección normal (promedio 59,7 por ciento) todos los sujetos estudiados tuvieron actividad de renina plasmática normal. Conclusión: No se detectó alteración en la actividad de renina plasmática en este grupo de pacientes con EPOC estable y sin antecedentes de edemas o disfunción ventricular izquierda. Para alcanzar el verdadero papel de las alteraciones neurohormonales en la fisiopatología de la EPOC es necesario estudiar sujetos con mayor grado de hipercapnia o con diferente función ventricular izquierda.

Purification and assay methods for angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE; EN 3.4.15.1) is a peptidyl dipeptide hydrolase that removes the carboxyl terminal His-Leu from angiotensin I to produce the octapeptide angiotensin II. In addition, ACE inactivates bradykinin, a vasodilator peptide/mediator of inflammation, as well as substance P, enkephalins and endorphins. Because of the importance of ACE and its active site-directed inhibitors in the pathogenesis and treatment of cardiovascular disorders such as hypertension and heart failure, ACE purification and assay are of clinical and commercial, as well as scientific interest. This review summarizes the historical development of ACE purification and assay methods and presents some innovative high-performance liquid chromatography-based techniques developed in our own laboratory for high yield and efficient purification and sensitive and specific assay of ACE.

Specific prorenin/renin binding (ProBP). Identification and characterization of a novel membrane site.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Recombinant human renin produced in different expression systems: biochemical properties and 3D structure.

Human renin has been expressed in Sf9 and CHO cells using two different gene constructs. The first construct contained a foreign signal peptide fused directly to the sequence encoding mature renin, whereas the second construct harbors the sequence for preprorenin. Prorenin was produced in significantly higher amounts than the mature enzyme expressed without its propeptide in both expression systems. Both directly expressed mature renin and proteolytically derived active renin have been purified and cocrystallized with the renin inhibitor Ro 42-5892. The 3D structure has been solved for both versions and demonstrates identity despite different glycosylation and different N termini.

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.

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum.

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37 degrees C, the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptide as products, with a specific activity of 1.35 pmol AI/min/mg (Km 22 microM; Kcat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC50 = 4.6 microM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.