Immunoreactive Renin Research Articles

Abstract 19: Effect of the endothelin receptor antagonist aprocitentan on plasma endothelin, renin and aldosterone concentrations in patients with resistant hypertension. A biomarker analysis of the PRECISION study.

Hypothesis: Dual endothelin (ET) ET A /ET B receptor antagonist aprocitentan significantly reduced blood pressure in patients with resistant hypertension, who were still hypertensive despite standard background anti-hypertensive drugs including a diuretic, an ARB and a CCB (PRECISION trial, NCT03541174). The effect of aprocitentan in this population, characterized with low renin and elevated aldosterone levels, was assessed on a panel of plasma biomarkers: ET-1, CT-proET-1 (a stable peptide and biomarker for ET-1 production), renin, aldosterone. Methods: From the 730 randomized patients in PRECISION, ET-1 (n=700), CT-proET-1 (n=100, randomly selected), immunoreactive renin (n=679) and aldosterone (n=703) were measured in plasma samples collected at randomization (baseline) and after 4 weeks for either placebo, aprocitentan 12.5 or 25 mg. Results: Four-week placebo treatment had no effect on the studied biomarkers compared to baseline. Treatment with aprocitentan 12.5 and 25 mg increased plasma ET-1 by +34% and +53% and CT-proET-1 by +38% and +47% vs. baseline respectively. Aprocitentan had no effect on renin levels (p>0.05) while decreasing plasma aldosterone by -17% and -24% at the 12.5 and 25 mg dose respectively. Importantly, the reduction in aldosterone did not result in a change in kalemia (see Table ). Conclusions: Aprocitentan caused reactive dose-dependent increases in plasma CT-proET-1 and ET-1, resulting from effective ET receptor blockade. The increase in CT-proET-1 may indicate a compensatory increase in ET-1 de novo synthesis, while the increase in ET-1 may reflect a reduced clearance due to blockade of ET B and increased production. The decrease of aldosterone by aprocitentan confirms the secretagogue role of ET-1 on aldosterone production by adrenal cortical cells via both ET A and ET B receptors This decrease of aldosterone was observed on top of the known decrease induced by an ARB but did not induce hyperkalemia nor aggravation of renal function.

SAT-058 Histopathological Analysis of Kidneys and Adrenal Glands in the Same Primary Aldosteronism (PA) Patients: Exploring the Mechanisms of Aldosterone Specific Renal Injuries

Primary aldosteronism (PA) is a major cause of secondary hypertension, accounting for 5 to 10% of all of the patients with hypertension. PA is also well known to harbor relatively higher incidence of cardiovascular complications than essential hypertension (EH) despite similar blood pressure levels. Among these complications, renal injuries are considered clinically important because up to 15% of APA (Aldosterone-producing adenoma) patients had already been chronic kidney disease at the time of its initial diagnosis (Iwakura et al., JCEM, 2014). In addition, PA patients are relatively younger than EH, which makes early clinical intervention pivotal. Many pathways of aldosterone-induced renal injuries have been proposed but the actual mechanism of aldosterone related injuries in human kidney has remained virtually unknown. Therefore, in this study, we performed the detailed histopathological analysis for kidneys and adrenal glands of the same PA patients and compared with those of EH. We analyzed 7 APA cases undergoing renal biopsy or nephrectomy with adrenalectomy. As a control group, 40 age-matched EH cases (31 autopsy, 9 biopsy) were examined. Digital image software (HALO, Indica Labs) was employed to perform quantitative histological analysis. There were no significant differences of eGFR between EH and PA before adrenalectomy (p=0.8111), but eGFR become lower in post-operative PA than in EH (p=0.0511). The prevalence of global glomerular sclerosis (GGS) and focal glomerular sclerosis (FGS) was significantly higher in PA than in EH (p=0.0031, 0.0035). Mesenchymal fibrosis was also more marked in PA than in EH (p=0.0091). Among the increased GGS groups (over 10%), the ratio of GGS/mesenchymal fibrosis tended to be lower in PA than in EH(p=0.0653). CYP11B2 positive area in adrenal cortex was more extended in autopsy EH than in adjacent cortex of PA (p=0.0209), and a significant correlation was also detected between CYP11B2 and renin immunoreactivity in autopsy EH adrenals (p=0.0067). In PA, CYP11B2 immunoreactivity in adenomas tended to be correlated with glomerular swelling (p=0.0539), probably reflecting the status of glomerular hyperfiltration. 11βHSD2 immunoreactivity of cortical tubules was significantly higher in PA than in EH (p=0.0001) and significantly correlated with the degree of renal arteriole hyalinization in PA (p<.0001). The renal injuries in PA demonstrated not only glomerular sclerosis but also mesenchymal fibrosis in contrast to kidneys of EH which predominantly demonstrated glomerular sclerosis. Higher 11βHSD2 expression in the tubules of PA kidneys could also enhance topical effects of aldosterone, resulting in renal arteriole hyalinization in PA. We firstly demonstrated the markedly different patterns of renal injuries between PA and EH patients and could contribute to the management of renal functions of these two different hypertensive disorders.

COX-2-derived PGE2 triggers hyperplastic renin expression and hyperreninemia in aldosterone synthase-deficient mice

Pharmacological inhibition or genetic loss of function defects of the renin angiotensin aldosterone system (RAAS) causes compensatory renin cell hyperplasia and hyperreninemia. The triggers for the compensatory stimulation of renin synthesis and secretion in this situation may be multimodal. Since cyclooxygenase-2 (COX-2) expression in the macula densa is frequently increased in states of a defective RAAS, we have investigated a potential role of COX-2 and its derived prostaglandins for renin expression and secretion in aldosterone synthase-deficient mice (AS−/−) as a model for a genetic defect of the RAAS. In comparison with wild-type mice (WT), AS−/− mice had 9-fold and 30-fold increases of renin mRNA and of plasma renin concentrations (PRC), respectively. Renin immunoreactivity in the kidney cortex of AS−/− mice was 10-fold higher than in WT. Macula densa COX-2 expression was 5-fold increased in AS−/− kidneys relative to WT kidneys. Treatment of AS−/− mice with the COX-2 inhibitor SC-236 for 1 week lowered both renal renin mRNA and PRC by 70%. Hyperplastic renin cells in AS−/− kidneys were found to express the prostaglandin E2 receptors EP2 and EP4. Global deletion of EP2 receptors did not alter renin mRNA nor PRC values in AS−/− mice. Renin cell-specific inducible deletion of the EP4 receptor lowered renin mRNA and PRC by 25% in AS−/− mice. Renin cell-specific inducible deletion of the EP4 receptor in combination with global deletion of the EP2 receptor lowered renin mRNA and PRC by 70–75% in AS−/− mice. Lineage tracing of renin-expressing cells revealed that deletion of EP2 and EP4 leads to a preferential downregulation of perivascular renin expression. Our findings suggest that increased macula densa COX-2 activity in AS−/− mice triggers perivascular renin expression and secretion via prostaglandin E2.

Bradykinin/B2 receptor activation regulates renin in M-1 cells via protein kinase C and nitric oxide.

In the collecting duct (CD), the interactions of renin angiotensin system (RAS) and kallikrein‐kinin system (KKS) modulate Na+ reabsorption, volume homeostasis, and blood pressure. In this study, we used a mouse kidney cortical CD cell line (M‐1 cells) to test the hypothesis that in the CD, the activation of bradykinin B2 receptor (B2R) increases renin synthesis and release. Physiological concentrations of bradykinin (BK) treatment of M‐1 cells increased renin mRNA and prorenin and renin protein contents in a dose‐dependent manner and increased threefold renin content in the cell culture media. These effects were mediated by protein kinase C (PKC) independently of protein kinase A (PKA) because B2R antagonism with Icatibant and PKC inhibition with calphostin C, prevented these responses, but PKA inhibition with H89 did not modify the effects elicited by the B2R activation. BK‐dependent stimulation of renin gene expression in CD cells also involved nitric oxide (NO) pathway because increased cGMP levels and inhibition of NO synthase with L‐NAME prevented it. Complementary renin immunohistochemical studies performed in kidneys from mice with conventional B2R knockout and conditional B2R knockout in the CD, showed marked decreased renin immunoreactivity in CD, regardless of the renin presence in juxtaglomerular cells in the knockout mice. These results indicate that the activation of B2R increases renin synthesis and release by the CD cells through PKC stimulation and NO release, which support further the interactions between the RAS and KKS.

Immunohistochemical localization of renin-containing cells in two elasmobranch species.

Renin immunoreactivity was localized at the light and electron microscopic level in two elasmobranch fish species, the Atlantic stingray, Dasyatis sabina, and river ray, Potamotrygon humerosa. At the light microscopic level, the peroxidase-anti-peroxidase method showed a positive immunoreactivity in modified smooth muscle cells in kidney afferent arterioles as well as in arterioles of several organs: rectal gland, inter-renal gland, conus arteriosus, and gill. Electron microscopic renin-positive immunogold localization was confined to the contents of membrane bound granules in the modified smooth muscle cells of these arterioles. The presence of renin-containing granules in the modified smooth muscle, "granular cells," of the renal glomerular afferent arteriole of these two stingray species adds support to earlier studies which showed the structural components of a complete juxtaglomerular apparatus and some of the biochemical and molecular components of a renin-angiotensin system (RAS) as found in teleost fish, reptiles, birds, and mammals. A notable result, however, was the renin-positive immunoreaction in the arteriolar wall of all other organs studied here. The presence of this "diffuse renin system" in the connective tissue of various organs suggests that in these two stingray species in addition to local organ-specific functions, the RAS may act as a systemic mechanism to regulate blood pressure and blood flow in the body.

Combination of LC–MS/MS aldosterone and automated direct renin in screening for primary aldosteronism

BackgroundAldosterone to renin ratio (ARR) is used in screening for primary aldosteronism (PA). However, there are only few studies on the influence of assay methods on ARR and its cut-off limits. MethodsPlasma direct renin immunoreactivity by chemiluminescence immunoassay (DR) was compared to renin activity assay (PRA), and a specific liquid chromatography–mass spectrometric method (LC–MS/MS) to radioimmunoassay (RIA) for plasma aldosterone. There were 75 samples for renin assays, and 42 samples of 39 patients for both renin and aldosterone assays. PA screening was considered positive if ARR by the aldosterone RIA:PRA was ≥800pmol/L:μg/L/h or by LC–MS/MS:DR≥44pmol/L:ng/L. ResultsThe correlation between the DR and PRA methods (n=75, r2=0.845) and between LC–MS/MS and RIA (n=42, r2=0.973) was high in general, but low between the renin methods (n=49, r2=0.435) at low PRA values. When ARR was used in screening for PA, there were three divergent cases (positive only by alternative methods), but when applied in combination with criteria for elevated aldosterone, the methods showed good agreement, resulting in eight positive and 31 negative screening results. ConclusionsThe automated DR assay combined with LC–MS/MS method for aldosterone provides a rapid, reliable, and specific method for screening of PA.

A rare cause of systemic hypertension in an African child: Questions

A female toddler was referred to our hospital with the problem of hypertensive encephalopathy manifesting with convulsions, right hemiparesis and blindness. She had been well until the age of 17 months when she developed fever and symptoms of an upper respiratory tract infection. Her past medical history was significant only for having been a small for gestational age baby. She weighed 2 kg at birth after a term, uncomplicated pregnancy. She had achieved her milestones normally up to the age of 12 months. Thereafter her mother noticed that her neurological development was faltering—she was not talking or playing, she was disinterested in her environment and she was unusually irritable. She has no family history of hypertension, heart or kidney disease. She has one healthy sibling. On physical examination she appeared chronically ill. She was severely stunted (length 72.5 cm, Z score<−3 SD) and wasted (weight for length Z score<−3 SD). Her level of consciousness was depressed. All her pulses were present and normal and there was no radio-femoral delay. Her blood pressure was 148/92 mm Hg and was elevated in all four limbs. She had puffy eyes, but no pedal or sacral oedema. She was clinically anaemic and mildly dehydrated. She did not have any lymphadenopathy. There were no cafe au lait spots, no malar rash and no evidence of vasculitis. The left precordium was bulging. The apex was heaving in the fifth intercostal space, displaced laterally. The second heart sound was loud. There was no murmur over the heart, the subclavian or carotid vessels, over the inter-scapular area or abdominal aorta. There was weakness of the left arm and leg, and all tendon reflexes were markedly brisk. She had complete loss of vision, but her other cranial nerves were intact. Fundoscopy revealed retinal haemorrhages, but no papilloedema. Laboratory investigations revealed the following information: she had a hypokalaemic metabolic alkalosis; potassium was 2.8 mmol/l and bicarbonate was 30 mmol/l. The serum sodium, chloride, urea and creatinine levels were normal. The full blood count showed a hypochromic microcytic anaemia, with haemoglobin of 9.4 g/dl [normal 11.1–14.7 g/dl, mean corpuscular volume of 65.9 fl (normal 70–86 fl) and mean corpuscular haematocrit of 20.8 pg (normal 23–31 pg)]. The differential white cell count was normal. Further investigations showed a suppressed plasma immuno-reactive renin of <1.8 mIU/l (normal 5.4–28.8 mIU/l) and aldosterone level of <31 pmol/l (normal 28–666 pmol/l). The urine potassium was 21.7 mmol/l, urine sodium was <10 mmol/l and urine chloride was 20 mmol/l. The urine protein creatinine ratio was 0.23 g/mmol (normal <0.02 g/mmol). Steroid chromatography excluded endocrine causes of hypertension associated with hypokalaemic metabolic alkalosis. The kidney sonography showed normal-sized kidneys for her length of 72.5 cm (right 6.1×3.1 cm, left 5.8×3 cm), with decreased cortico-medullary differentiation. Renal The answers to these questions can be found at http://dx.doi.org/10.1007/ s00467-013-2446-3

New Renin Inhibitor VTP-27999 Alters Renin Immunoreactivity and Does Not Unfold Prorenin

Renin inhibitors like aliskiren not only block renin but also bind prorenin, thereby inducing a conformational change (like the change induced by acid) allowing its recognition in a renin-specific assay. Consequently, aliskiren can be used to measure prorenin. VTP-27999 is a new renin inhibitor with an aliskiren-like IC50 and t1/2, and a much higher bioavailability. This study addressed (pro)renin changes during treatment of volunteers with VTP-27999 or aliskiren. Both drugs increased renin immunoreactivity. Treatment of plasma samples from aliskiren-treated subjects with excess aliskiren yielded higher renin immunoreactivity levels, confirming the presence of prorenin. Unexpectedly, this approach did not work in VTP-27999-treated subjects, although an assay detecting the prosegment revealed that their blood still contained prorenin. Subsequent in vitro analysis showed that VTP-27999 increased renin immunoreactivity for a given amount of renin by ≥ 30% but did not unfold prorenin. Yet, it did bind to acid-activated, intact prorenin and then again increased immunoreactivity in a renin assay. However, no such increase in immunoreactivity was seen when measuring acid-activated prorenin bound to VTP-27999 with a prosegment-directed assay. The VTP-27999-induced rises in renin immunoreactivity could be competitively prevented by aliskiren, and antibody displacement studies revealed a higher affinity of the active site-directed antibodies in the presence of VTP-27999. In conclusion, VTP-27999 increases renin immunoreactivity in renin immunoassays because it affects the affinity of the active site-directed antibody. Combined with its lack of effect on prorenin, these data show that VTP-27999 differs from aliskiren. The clinical relevance of these results needs to be established.

Long‐Term Antihypertensive Effects of Aliskiren, a Direct Renin Inhibitor, in Chronic Hemodialysis Patients

The renin-angiotensin-aldosterone system is not necessarily suppressed in end-stage renal disease patients undergoing dialysis. Of all the inhibitors of this system, the clinical efficacy of the renin inhibitor, aliskiren, has not been well demonstrated in dialysis patients. We evaluated the antihypertensive effect of aliskiren, administered as a single daily dose of 150 mg for 24 weeks, in 23 chronic hemodialysis patients (age 65 ± 12 years, 15 men and eight women) with blood pressure ≥140/90 mm Hg, and assessed the factors relating to blood pressure reduction. At 4 weeks, the average systolic blood pressure before the dialysis session was insignificantly reduced from 163 ± 10 mm Hg to 160 ± 15 mm Hg, while it was significantly lowered at 12 (154 ± 13 mm Hg) and 24 weeks (155 ± 10 mm Hg), although the pulse rate was not significantly altered. Serum K increased at 24 weeks from 4.9 ± 0.6 mEq/L to 5.2 ± 0.8 mEq/L. Only 10 out of 23 patients showed systolic blood pressure reduction by ≥10 mm Hg. Naturally, plasma renin immunoreactivity increased, while plasma renin activity, along with angiotensin II and aldosterone levels decreased. Basal levels of the components of the renin-angiotensin-aldosterone system were not significantly different in patients showing systolic blood pressure reduction by ≥10 mm Hg (n = 10) vs. those with <10 mm Hg changes (n = 13). The reduction in systolic blood pressure in all 23 patients taken as a whole correlated with changes in plasma renin activity (r = -0.432, P < 0.05) and angiotensin II (r = 0.467, P < 0.05). In chronic hemodialysis patients, aliskiren modestly lowers blood pressure over the long term, although the antihypertensive effect seems dependent on the changes, but not on the basal levels of plasma renin activity and angiotensin II.

Activation of the renin-angiotensin system by a low-salt diet does not augment intratubular angiotensinogen and angiotensin II in rats

In angiotensin II (ANG II) infusion hypertension, there is an augmentation of intratubular angiotensinogen (AGT) and ANG II leading to increased urinary AGT and ANG II excretion rates associated with tissue injury. However, the changes in urinary AGT and ANG II excretion rates and markers of renal injury during physiologically induced stimulation of the renin-angiotensin system (RAS) by a low-salt diet remain unclear. Male Sprague-Dawley rats received a low-salt diet (0.03% NaCl; n = 6) and normal-salt diet (0.3% NaCl, n = 6) for 13 days. Low-salt diet rats had markedly higher plasma renin activity and plasma ANG II levels. Kidney cortex renin mRNA, kidney AGT mRNA, and AGT immunoreactivity were not different; however, medullary renin mRNA, kidney renin content, and kidney ANG II levels were significantly elevated by the low-salt diet. Kidney renin immunoreactivity was also markedly increased in juxtaglomerular apparati and in cortical and medullary collecting ducts. Urinary AGT excretion rates and urinary ANG II excretion rates were not augmented by the low-salt diet. The low-salt diet caused mild renal fibrosis in glomeruli and the tubulointerstitium, but no other signs of kidney injury were evident. These results indicate that, in contrast to the response in ANG II infusion hypertension, the elevated plasma and intrarenal ANG II levels caused by physiological stimulation of RAS are not reflected by increased urinary AGT or ANG II excretion rates or the development of renal injury.

Two-site automated chemiluminescent assay for measurement of immunoreactive renin

Measurement of renin is important for the clinical assessment of hypertensive patients and for the screening for primary aldosteronism. The aim of this study was to evaluate the performances of an automated immunoassay for measurement of immunoreactive renin. Functional sensitivity, in vitro stability, and reference values were determined. Method comparison with the plasma renin activity assay was also performed. Our results demonstrate that the Liaison® direct renin assay may assist the clinician in the assessment of hypertensive patients and in the screening for primary aldosteronism.

Proteinase-Activated Receptors 1 and 2 Exert Opposite Effects on Renal Renin Release

Proteinase-activated receptors (PARs) 1 to 4 are highly expressed in the kidney and are involved in the regulation of renal hemodynamics and tubular function. Since intravascular infusion of the proteinase thrombin, which activates PARs, has been shown to decrease plasma renin activity in rats, we investigated the effects of the respective PAR subtypes on renin release using the isolated perfused mouse kidney model. Thrombin dose-dependently reduced perfusate flow and inhibited renin secretion rates (RSRs) that had been prestimulated by the β-adrenoreceptor agonist isoproterenol. The suppression of RSRs was prevented by the selective PAR1 inhibitor SCH79797, and direct activation of PAR1 by TFLLR mimicked the effects of thrombin on RSRs and vascular tone. Moreover, TFLLR suppressed the stimulations of RSRs in response to the loop diuretic bumetanide, to prostaglandin E(2), or to a decrease in renal perfusion pressure but not in response to a reduction in extracellular calcium. The PAR2-activating peptide SLIGRL concentration dependently increased RSR and perfusate flow. The stimulation of RSRs by SLIGRL was markedly attenuated by N(G)-nitro-L-arginine methyl ester, suggesting an NO-dependent mechanism. Activation of PAR4 by AYPGKF did not modulate RSRs or perfusate flow. PAR1 and PAR2 immunoreactivity were detected in the juxtaglomerular region and were colocalized with renin immunoreactivity. Our data provide evidence that PAR1 activation inhibits renal renin secretion and induces renal vasoconstriction, whereas PAR2 activation stimulates renin release and induces vasodilation mainly via the release of NO.

Transcriptional regulator RBP-J regulates the number and plasticity of renin cells

Renin-expressing cells are crucial in the control of blood pressure and fluid-electrolyte homeostasis. Notch receptors convey cell-cell signals that may regulate the renin cell phenotype. Because the common downstream effector for all Notch receptors is the transcription factor RBP-J, we used a conditional knockout approach to delete RBP-J in cells of the renin lineage. The resultant RBP-J conditional knockout (cKO) mice displayed a severe reduction in the number of renin-positive juxtaglomerular apparatuses (JGA) and a reduction in the total number of renin positive cells per JGA and along the afferent arterioles. This reduction in renin protein was accompanied by a decrease in renin mRNA expression, decreased circulating renin, and low blood pressure. To investigate whether deletion of RBP-J altered the ability of mice to increase the number of renin cells normally elicited by a physiological threat, we treated RBP-J cKO mice with captopril and sodium depletion for 10 days. The resultant treated RBP-J cKO mice had a 65% reduction in renin mRNA levels (compared with treated controls) and were unable to increase circulating renin. Although these mice attempted to increase the number of renin cells, the cells were unusually thin and had few granules and barely detectable amounts of immunoreactive renin. As a consequence, the cells were incapable of fully adopting the endocrine phenotype of a renin cell. We conclude that RBP-J is required to maintain basal renin expression and the ability of smooth muscle cells along the kidney vasculature to regain the renin phenotype, a fundamental mechanism to preserve homeostasis.

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin

Renin expression in adult renal epithelial tumors with granular cells

Renal cell tumors have been shown to be associated with secretory products, including renin, but the frequency of renin expression is not known. To investigate renin expression in epithelial renal neoplasms, we examined a series of 89 adult renal tumors with granular cells using a monoclonal antiserum to detect the hormone by immunohistochemistry (IHC) and their association with hypertension. We found that 25 of 89 tumors (28.1%) contained immunoreactivity for renin. Oncocytomas had the highest percentage of cases with renin expression: 8 of 13 (61.5%), all of these with diffuse immunostaining. Renin was detected in 31.6% of 38 chromophobe carcinomas and in 12.5% of 24 conventional carcinomas. Renin was not detected in collecting duct carcinomas or in mucinous tubular and spindle cell carcinomas. Systemic hypertension was detected in 11 of 25 (44%) patients with renin expression and in 32 of 64 (50%) patients without renin immunolabeling ( p = 0.64). After tumor resection, patients with renin expression and high blood pressure showed no hypertension remission. In conclusion, renin is frequently expressed in renal epithelial neoplasms with granular cells, mainly in oncocytomas, but this renin appears clinically inactive.

Detección de renina en carcinomas cromófobos de células renales

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 χ 2 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.

A comparative study on inter and intralaboratory reproducibility of renin measurement with a conventional enzymatic method and a new chemiluminescent assay of immunoreactive renin

The activity of the renin-angiotensin system is usually evaluated as plasma renin activity (PRA, ngAI/ml per h) but the reproducibility of this enzymatic assay is notoriously scarce. We compared the inter and intralaboratory reproducibilities of PRA with those of a new automated chemiluminescent assay, which allows the direct quantification of immunoreactive renin [chemiluminescent immunoreactive renin (CLIR), microU/ml]. Aliquots from six pool plasmas of patients with very low to very high PRA levels were measured in 12 centres with both the enzymatic and the direct assays. The same methods were applied to three control plasma preparations with known renin content. In pool plasmas, mean PRA values ranged from 0.14 +/- 0.08 to 18.9 +/- 4.1 ngAI/ml per h, whereas those of CLIR ranged from 4.2 +/- 1.7 to 436 +/- 47 microU/ml. In control plasmas, mean values of PRA and of CLIR were always within the expected range. Overall, there was a significant correlation between the two methods (r = 0.73, P < 0.01). Similar correlations were found in plasmas subdivided in those with low, intermediate and high PRA. However, the coefficients of variation among laboratories found for PRA were always higher than those of CLIR, ranging from 59.4 to 17.1% for PRA, and from 41.0 to 10.7% for CLIR (P < 0.01). Also, the mean intralaboratory variability was higher for PRA than for CLIR, being respectively, 8.5 and 4.5% (P < 0.01). The measurement of renin with the chemiluminescent method is a reliable alternative to PRA, having the advantage of a superior inter and intralaboratory reproducibility.

BRIEF CRYOACTIVATION MARKEDLY AFFECTS PLASMA RENIN ACTIVITY AND DIRECT RENIN MEASUREMENT IN LOW RENIN SAMPLES: PP.24.473

Background: Exposure of plasma to low temperature (0°-4°C) i.e. cryoactivation (C) is known to increase plasma renin activity (PRA, ng/ml/h) but it is not known to what extent C affects the measurement of immunoreactive renin (IR, mU/L) measured with the direct renin assays (DRA) in plasmas with different renin content. Methods: Aliquots of human pool plasmas with low (L), intermediate (I) and high (H) renin were exposed to C for 0, 24, 48, 72 and 96 hours (hrs) and then measured in triplicate as PRA with the conventional enzymatic method and as IR with a new chemiluminescent DRA (Dia-Sorin LIAISON). Results: In L samples PRA rose from 0.14 ± 0.01at baseline to 0.53 ± 0.01(+ 270%) and to 1.1 ± 0.03 (+ 680%) respectively after 24 and 96 hrs of C whereas at the corresponding times IR rose from a baseline of 4.1 ± 0.1 to 11.3 ± 0.4 (+ 174%) and to 24.6 ± 0.4 (+ 595%). In I samples, for the same hrs of C, PRA rose from 1.9 ± 0.02 to 2.3 ± 0.04 (+ 17%) and to 4.1 ± 0.05 (+ 110%), whereas IR rose from 48.7 ± 0.3 to 61.7 ± 0.9 (+ 27%) and to 118 ± 3 (+ 142%). In H samples PRA rose from 7.8 ± 0.2 at baseline to 8.7 ± 0.2 (+ 11%) and to 10.5 ± 0.6 (+ 34%) whereas IR rose from 222 ± 2 to 241 ± 2 (8%) and to 289 ± 2 (+ 30%). The increments of PRA and IR were always statistically significant with respect to baseline values and, in percent, similar with the two assays. Conclusions: Thus C affects the measurement of renin to the same extent with the two assays, but the percent increases in PRA and IR are particularly relevant in L samples. In these cases even short period of inadvertent C can lead to an overestimation of the true renin levels.

CONFORMATIONAL CHANGES IN PRORENIN DURING RENIN INHIBITION IN VITRO AND IN VIVO AS ASSESSED BY A CHEMOLUMINESCENCE IMMUNOASSAY: PP.24.469

We have previously shown that the renin inhibitor, aliskiren, induce changes in the conformation of prorenin in vitro and influence the quantification of active renin by the Cisbio and the Nichols immunoradiometric assays. The difference between the two assays depended on incubation time in vitro. Objective: to test whether such phenomenon occurs with a shorter incubation time (30–40 min) using the chemoluminescence DiaSorin Liaison® immunoassay by comparison to the Cisbio assay (3 h incubation). Methods: Immunoreactive active renin concentrations were measured 1) in plasma samples taken from 5 healthy subjects incubated with aliskiren 10 nM to 1 mM in vitro for < 1 h and 24 h (DiaSorin) and for 3 h and 24 h (Cisbio), and 2) in plasma samples collected in a single oral dose crossover study comparing aliskiren (300 mg) and valsartan (160 mg), in 16 normotensive subjects. Results: Aliskiren added to plasma samples in vitro increased active renin immunoreactivity in both assays. The increase was significant from 10 μM aliskiren onwards (+107% [95%CI:39;208] in the DiaSorin assay and +183% [95%CI:42;465] in the Cisbio assay, p < 0.0001) and reached +144% (95%CI:64;263) and +246.8% (95%CI:74;592) at 1 mM aliskiren, respectively. 24 h incubation amplified the magnitude of the phenomenon which started to be significant at lower aliskiren concentration (100 nM) in both assays. In the crossover study, the two assays gave similar values for plasma active renin concentration before treatment and following valsartan administration (intraclass coefficient for agreement between the 2 assays:0.84 [95%CI:0.60;0.94]). However, a Bland-Altman plot showed a systematic bias towards higher values (1.76 times higher [95% CI:1.66;1.87]) in the DiaSorin than in the Cisbio assay following aliskiren administration. Conclusion: Even with a short incubation time, aliskiren at high concentration interfere with the recognition of active renin molecules by the monoclonal antibodies used in the DiaSorin assay. Careful consideration must be given to the methodology used for quantifying immunoreactive plasma active renin when patients are treated with aliskiren.

Detection of renin in chromophobe renal cell carcinomas

AimTo evaluate frequency of renin detection in chromophobe renal cell carcinoma, and if this expression was associated to systemic high blood pressure. Material and methodsA 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. ResultsWe 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 no patient with renin expression and high blood pressure showed remission of the hypertension. ConclusionRenin 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.