Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension

Plasma renin activity (PRA) is an essential analytical tool for screening and diagnosis of secondary forms of hypertension. Typically, PRA is measured by competitive radioimmunoassay, but there are significant drawbacks to this technique including non-specificity, long analysis times, narrow calibration range, and the requirement for radionucleotides. In this paper, we report a method for plasma renin activity determination by immuno-MALDI mass spectrometry detection. This method overcomes the issues of non-specificity and long analytical times present with RIA, and does not require the use of radionucleotides. As an initial methodological evaluation, plasma renin activity results obtained by radioimmunoassay, LC/ESI-MS/MS, and immuno-MALDI on 64 samples from an outpatient primary aldosteronism screening program have been compared. A strong correlation was found between immuno-MALDI and radioimmunoassay (R² = 0.9412, 62/64 within the 95% CI of the Bland-Altman plot), and iMALDI and LC/ESI-MS/MS (R² = 0.9471, 62/64 within the 95% CI of the Bland-Altman plot). Technical replicates showed a 4.8% CV, while inter- and intra-day replicates showed CVs of 17.3% and 17.2% respectively. We have developed an assay capable of measuring PRA without the use of radionucleotides. This immuno-MALDI approach affords the specificity of MS while avoiding the long analytical run times and technical problems associated with HPLC. With the use of robotic sample preparation to optimize precision, this assay should be adaptable to clinical environments.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

Synthesis and properties of the first all-aza analogue of a biologically active peptide

The synthesis of the first all-aza-amino acid analogue ( 2 ) of a peptidic renin inhibitor is described. The X-ray structural analysis and molecular modelling investigations of this novel compound reveal interesting conformational features which have a significant impact on its biological activity. In addition, insight into conformational features of azapeptides in general in comparison with the corresponding purely peptidic compounds is given.     

6周大强度训练对大鼠肾功能的影响及其机制

目的：研究6周大强度训练对大鼠肾功能影响及运动性蛋白尿的机制。方法：6周龄SD雄性大鼠36只随机分为：安静对照组（C组,n=12）和大强度训练组（M组,n=24）。大鼠适应性饲养4 d后,C组不进行任何运动,M组采用6周递增负荷游泳训练。每周训练6 d,每天1次。第4周起开始负重（体重的1%）并逐渐递增（体重的6%）。各组大鼠末次训练结束后30 min取单次尿,24 h后腹主静脉取血,取双侧肾脏待测。HE染色观察肾脏肾小球结构,酶联免疫吸附法测定尿总蛋白、尿微量白蛋白、中性粒细胞明胶酶相关脂质运载蛋白,碱性苦味酸法测试尿肌酐,比色法测定血清肌酐、尿素氮,蛋白质免疫印迹法检测肾组织Nephrin蛋白表达量,放射免疫法测试血清睾酮、皮质酮和肾组织及血液中肾素-血管紧张素系统相关指标变化。结果：与C组比较,M组血清睾酮/皮质酮显著降低（P<0.01）;尿液蛋白总量、微量白蛋白、微量白蛋白与肌酐比值、中性粒细胞明胶酶相关脂质运载蛋白、血清尿素氮、肌酐均显著升高（P<0.01）;肾小球结构明显改变,且Paller评分明显增高（P<0.01）;肾组织Nephrin蛋白表达量明显降低（P<0.01）;肾脏内部与循环血液中肾素活性、血管紧张素Ⅱ显著增高（P<0.01）。结论：6周大强度训练对大鼠肾功能影响及运动性蛋白尿的机制可能为过度训练诱导肾脏内部及循环系统中肾素-血管紧张素系统持续兴奋,并下调Nephrin蛋白的表达,进而导致肾脏结构与功能异常,出现蛋白尿。     

Perinatal α-tocopherol overload programs alterations in kidney development and renal angiotensin II signaling pathways at birth and at juvenile age: Mechanisms underlying the development of elevated blood pressure

α-Tocopherol (α-Toc) overload increases the risk of dying in humans (E.R. Miller III et al. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality Ann Int Med. 142 (2005) 37–46), and overload during early development leads to elevation of blood pressure at adult life, but the mechanism(s) remains unknown. We hypothesized that α-Toc overload during organogenesis affects the renal renin angiotensin system (RAS) components and renal Na⁺ handling, culminating with late elevated blood pressure. Pregnant Wistar rats received α-Toc or the superoxide dismutase mimetic tempol throughout pregnancy. We evaluated components of the intrarenal renin angiotensin system in neonate and juvenile offspring: Ang II-positive cells, Ang II receptors (AT₁ and AT₂), linked protein kinases, O₂^? production, NADPH oxidase abundance, lipid peroxidation and activity of Na⁺-transporting ATPases. In juvenile offspring we followed the evolution of arterial blood pressure. Neonates from α-Toc and tempol mothers presented with accentuated retardment in tubular development, pronounced decrease in glomerular Ang II-positive cells and AT₁/AT₂ ratio, intense production of O₂^? and upregulation of the α, ε and λ PKC isoforms. α-Toc decreased or augmented the abundance of renal (Na⁺+K⁺)ATPase depending on the age and α-Toc dose. In juvenile rats the number of Ang II-positive cells returned to control values as well as PKCα, but co-existing with marked upregulation in the activity of (Na⁺+K⁺) and Na⁺-ATPase and elevated arterial pressure at 30?days. We conclude that the mechanisms of these alterations rely on selective targeting of renal RAS components through genic and pro-oxidant effects of the vitamin.     

Selected zinc metabolism parameters in premenopausal and postmenopausal women with moderate and severe primary arterial hypertension

The aim of this study was to compare zinc (Zn) metabolism parameters in groups of premenopausal and postmenopausal women with moderate and severe primary arterial hypertension. The study included 38 women, of which 15 were premenopausal and 23 were postmenopausal. Postmenopausal women had a positive correlation between total (ERCt-Zn) and oubain-dependent (ERCos-Zn) rate constants of Zn efflux from lymphocyte (k = 0.52). In premenopausal women’s ERCos-Zn was negatively but weakly correlated with serum Zn (Zn-s) (k = 0.35). The Zn ERCt-Zn and ERCos-Zn did not show any correlation with age, as did Zn-s. Lymphocyte Zn correlated negatively with age only in premenopausal women (k = -0.62). The renin-angiotensin-aldosterone system correlated with Zn metabolism parameters. In premenopausal women, plasma renin activity and serum aldosterone showed positive correlations with lymphocyte Zn (Zn-l) (k = 0.63 andk = 0.41, respectively), and in postmenopausal women, it correlated negatively with Zn-s (k = -0.38) and whole aldosterone correlated negatively with ERCos-Zn (k = -0.41). Positive correlations between Zn metabolism parameters and arterial blood pressure in premenopausal women were as follows: ERCt-Zn with diastolic blood pressure (dRR) (k = 0.40) and ERCos-Zn with dRR (k = 0.47). In postmenopausal women, the correlations between ERC-t-Zn and dRR and systolic blood pressure (sRR) were negative (k = -0.53 andk = -0.63, respectively). A similar situation was observed between dRR and sRR and Zn-s (k = -0.40 andk = -0.38, respectively). The body mass index (BMI) was positively correlated with ERCt-Zn in premenopausal women (k = 0.36), whereas in postmenopausal, it was negatively correlated with ERCos-Zn (k = -0.42). For the whole group, negative correlations were seen between ZnS and dRR and sRR (k = -0.36 andk = -0.39, respectively) and between ERCos-Zn and BMI (k = -0.39). The results presented show differences in Zn metabolism in arterial hypertension between premenopausal and post-menopausal women. The role of estrogens in these differences is disscused.     

Modulation of renin angiotensin system components by high glucose levels in the culture of collecting duct cells

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin–angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D -glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1–7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1–7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance.     

Decrease of Choline Acetyltransferase Activity of Rat Cortex Capillaries with Aging

Choline acetyltransferase (ChAT) activity was estimated in brain cortex capillaries isolated from 3-, 12-, 18-, and 24-month-old rats. Maximum enzymatic activity was found at 12 months (55 +/- 0.3 pmol X mg-1 protein X min-1; mean +/- SEM) and then it decreased to reach a minimum at 24 months (34 +/- 3.1 pmol X mg-1 protein X min-1). A less marked decrease of enzymatic activity was also found in cortex homogenate and in a synaptosomal fraction obtained from the same groups of rats. Loss of ChAT of brain capillaries with aging could be related to a general phenomenon of cortical cholinergic deficit in that condition.     

Plasma catecholamines in man are not influenced by the inhibition of prostaglandin synthesis

Indomethacin has been reported to potentiate the release of noradrenaline from sympathetic nerve endings $\text{in vitro}$ and to increase urinary noradrenaline excretion in rats. We have studied the influence of indomethacin on plasma catecholamine levels in 10 normal men, using measurement of plasma renin activity (PRA) as an index of the pharmacodynamic effect of indomethacin. Both in the supine and standing positions indomethacin failed to alter the plasma concentrations of noradrenaline, adrenaline or dopamine, while PRA was markedly suppressed. It is concluded that in the intact human indomethacin does not influence catecholamine concentrations.