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

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

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.

A primitive type of renin-expressing lymphocyte protects the organism against infections.

The hormone renin plays a crucial role in the regulation of blood pressure and fluid-electrolyte homeostasis. Normally, renin is synthesized by juxtaglomerular (JG) cells, a specialized group of myoepithelial cells located near the entrance to the kidney glomeruli. In response to low blood pressure and/or a decrease in extracellular fluid volume (as it occurs during dehydration, hypotension, or septic shock) JG cells respond by releasing renin to the circulation to reestablish homeostasis. Interestingly, renin-expressing cells also exist outside of the kidney, where their function has remained a mystery. We discovered a unique type of renin-expressing B-1 lymphocyte that may have unrecognized roles in defending the organism against infections. These cells synthesize renin, entrap and phagocyte bacteria and control bacterial growth. The ability of renin-bearing lymphocytes to control infections-which is enhanced by the presence of renin-adds a novel, previously unsuspected dimension to the defense role of renin-expressing cells, linking the endocrine control of circulatory homeostasis with the immune control of infections to ensure survival.

Vasculometabolic and Inflammatory Effects of Aldosterone in Obesity.

CONTEXT: Not all obese individuals develop cardiovascular disease (CVD). Hyperaldosteronism is suggested to cause inflammation and metabolic dysregulation, and might contribute to CVD development in obese individuals. OBJECTIVE: We aimed to investigate the association of aldosterone concentrations with inflammation, metabolic disturbances, and atherosclerosis in overweight and obese individuals. Additionally, we measured renin concentrations to investigate whether the observed effects reflected general activation of the renin-angiotensin-aldosterone system (RAAS). DESIGN: A cross-sectional cohort study (300-OB study) was conducted. Various inflammatory parameters, traits of the metabolic syndrome, lipidome and metabolome parameters, fat distribution, and carotid atherosclerosis were associated with plasma aldosterone and renin levels. SETTING: The setting of this study was the Radboudumc (i.o. Radboudumc), the Netherlands. PATIENTS: A total of 302 individuals with a body mass index greater than or equal to 27 kg/m2 participated. MAIN OUTCOME MEASURES AND RESULTS: Aldosterone was associated with various markers of inflammation and metabolic dysregulation, which partly differed from the associations observed for renin. Although both were associated with inflammatory cell numbers, only renin was associated with classical markers of systemic inflammation. Both were associated with the metabolic syndrome and hepatic steatosis. Of the traits that constitute metabolic syndrome, aldosterone, but not renin, was associated with triglyceride concentrations. Accordingly, aldosterone was associated with large very low-density lipoprotein particles; metabolomics studies further associated aldosterone with urate concentrations and derivatives of the linoleic acid metabolism pathway. Neither aldosterone nor renin was associated with atherosclerotic plaque thickness. CONCLUSIONS: Aldosterone is not an important driver of systemic inflammation in the obese, whereas aldosterone concentrations and metabolic dysregulation are strongly intertwined in these individuals. Although prospective studies are necessary to validate these results, the independent effects of aldosterone on carotid atherosclerosis appear modest.

Renin-specific antibody for study of cardiovascular homeostasis.

Antiserum specific for purified canine renal renin was used to inhibit this enzyme in trained, conscious dogs. The antiserum did not affect blood pressure in sodium-replete dogs but decreased plasma renin activity and blood pressure in sodium-depleted animals. The antiserum also reduced blood pressure to control levels concomitant with suppression of plasma renin activity in uninephrectomized dogs with acute renovascular hypertension. These observations establish the role of the renin-angiotensin system in the maintenance of blood pressure in the sodium-depleted state as well as in the initiation of renovascular hypertension.

Immune Dysfunction and Risk of Infection in Chronic Kidney Disease.

Cardiovascular disease and infections are directly or indirectly associated with an altered immune response, which leads to a high incidence of morbidity and mortality, and together, they account for up to 70% of all deaths among patients with chronic kidney dysfunction. Impairment of the normal reaction of the innate and adaptive immune systems in chronic kidney disease predisposes patients to an increased risk of infections, virus-associated cancers, and a diminished vaccine response. On the other hand, an abnormal, exaggerated reaction of the immune systems can also occur in this group of patients, resulting in increased production and decreased clearance of proinflammatory cytokines, which can lead to inflammation and its sequelae (eg, atherosclerotic cardiovascular disease). Epigenetically, modifications in hematopoietic stem cells involving a shift from lymphoid to myeloid cell lineage may underlie uremia-associated immunological senescence, which is not reversed by renal replacement therapy, including kidney transplantation. Measures aimed at attenuating the immune abnormalities in chronic kidney disease/end-stage renal disease should be an area of focused research as this could potentially lead to a better understanding and, thus, development of therapies that could reduce the disastrously high death rate in this patient population. The aim of the present article is to review the characteristics, causes, and mechanisms of the immune dysfunction related to chronic kidney disease.

The renin-angiotensin system and therapeutic vaccines for hypertension.

With increased understanding of the pharmacology of the renin-angiotensin system (RAS), many researchers have explored immunological approaches to inhibit components of the RAS for the treatment of hypertension. Active and passive immunizations of the various components of the RAS have been performed, utilizing renin, angiotensin I, angiotensin II and angiotensin II receptor type 1 vaccines. This review discusses the RAS as a target for the development of a hypertension vaccine, and evaluates the safety and efficacy of these vaccines.

Immediate hypersensitivity elicits renin release from cardiac mast cells.

BACKGROUND: We recently reported that murine and cavian heart mast cells are a unique extrarenal source of renin. Ischemia/reperfusion releases this renin leading to local angiotensin formation and norepinephrine release. As mast cells are a primary target of hypersensitivity, we assessed whether anaphylactic mast cell degranulation also results in renin and norepinephrine release. METHODS: Hearts isolated from presensitized guinea pigs were challenged with antigen. RESULTS: Cardiac anaphylaxis was characterized by mast cell degranulation, evidenced by beta-hexosaminidase release and associated with renin and norepinephrine release. Mast cell stabilization with cromolyn or lodoxamide markedly attenuated the release of beta-hexosaminidase, renin and norepinephrine. Renin inhibition with BILA2157 did not affect mast cell degranulation, but attenuated norepinephrine release. CONCLUSIONS: Our findings disclose that immediate-type hypersensitivity elicits renin release from mast cells, activating a local renin-angiotensin system, thereby promoting norepinephrine release. As renin is stored in human heart mast cells, allergic reactions could initiate renin release, leading to local angiotensin formation and hyperadrenergic dysfunction.

Vaccination: a novel strategy for inhibiting the renin-angiotensin-aldosterone system.

Immunologic approaches to renin-angiotensin-aldosterone system (RAAS) inhibition have been studied for more than 50 years. In animal models, vaccination against renin was effective but resulted in fatal autoimmune renal disease; vaccines directed at small peptides including angiotensin I and II and a segment of the AT(1) receptor reduced blood pressure (BP) without causing autoimmune disease. In humans, angiotensin I vaccination did not reduce BP. More promising is the AngQb vaccine, which uses an immunization technology involving conjugation of angiotensin II to virus-like particles. In a phase 2 trial, hypertensive patients vaccinated with 300 microg showed a difference of 9.0/4.0 mm Hg from baseline in mean daytime ambulatory BP after 14 weeks (P = 0.015 for systolic BP, P = 0.064 for diastolic BP), and a marked reduction in early morning BP. No serious adverse events were attributed to vaccine administration. Although questions remain regarding efficacy and safety, RAAS immunization represents an innovative and promising approach to hypertension treatment.

[Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme].

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

Unexpected role of the human cytomegalovirus contribute to essential hypertension in the Kazakh Chinese population of Xinjiang.

Human cytomegalovirus (HCMV) infection, chronic inflammation and oxidative stress, the renin-angiotensin system (RAS), endothelial function, and DNA methylation play roles in the pathogenesis of essential hypertension (EH); however, the mechanism by which HCMV predisposes patients to hypertension remain unclear. Our group previously demonstrated an association between EH and HCMV infection in Kazakh Chinese. Here, we investigated the relationship between HCMV infection and other clinicopathological features in 720 Kazakh individuals with or without hypertension (n=360 each; age: 18-80). Multiple linear and logistic regression analyses were used to determine the associations between HCMV infection, clinical characteristics, and EH. Notably, patients with EH, particularly those with HCMV infection, exhibited a marked increase in tumor necrosis factor-α (TNF-α) and 8-hydroxy-2-deoxyguanosine (8-OHDG) levels, but a decrease in endothelial nitric oxide synthase (eNOS) and renin levels. Similarly, elevated TNF-α and 8-OHDG levels were independent predictors of increased HCMV antibody titers, whereas eNOS and renin were negatively correlated with the latter. Moreover, serum angiotensin-converting enzyme (sACE, ACE) methylation was increased, whereas 11-ß hydroxysteroid dehydrogenase 2 (HSD11ß2; HSD3B2) methylation was decreased in patients with EH who were also infected with HCMV. A positive correlation between HSD3B2 methylation and HCMV IgG titer and blood pressure was additionally observed, whereas angiotensin-converting enzyme (ACE) methylation was inversely correlated with blood pressure. Collectively, these data indicate that HCMV may contribute to EH development in the Kazakh Chinese by increasing TNF-α and 8-OHDG levels, suppressing eNOS and renin, and manipulating HSD3B2 and ACE methylation.

Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells.

Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin.

Renin biosynthesis by human tumoral juxtaglomerular cells. Evidences for a renin precursor.

Renin biosynthesis was studied in a juxtaglomerular cell tumor. The tumoral tissue had a high renin content (180 Goldblatt Units/g of tissue), was heavily stained by immunofluorescence using human renin antiserum, and exhibited numerous characteristic secretory granules by electron microscopy. In one series of experiments, renin biosynthesis was studied in tissue slices, by following the incorporation of radiolabeled amino acids into specific immunoprecipitable renin. Time course studies showed that renin was first synthesized in a high molecular weight form, 55,000 mol wt, i.e., 10,000 mol wt higher than that of active renin, and was then converted into a 44,000-mol wt form. In a second series of experiments renin tumoral cells were cultured. Small, round, birefringent cells obtained after collagenase digestion produced renin in both primary culture and subculture media. After 5 d most of the renin found in the culture medium was inactive, but could be activated by trypsin treatment. The tumoral tissue exhibited a strong renin immunofluorescence and numerous secretory granules were observed by electron microscopy. In contrast, the renin-producing cells isolated from this tumor and grown in culture showed little renin immunofluorescence and no secretory granule could be observed. The renin-producing cells in primary culture and subculture were pulsed with radiolabeled amino acids, and immunoprecipitable radiolabeled renin was found in the culture media, thus demonstrating the actual biosynthesis of the enzyme. This renin was not stored inside cultured cells but was rapidly released into the medium and had a molecular weight of 55,000. No conversion of this inactive high molecular weight renin into the active, 44,000 mol wt form of renin was observed. We postulate the existence of two pathways for the processing, packaging, and secretion of renin in the tumoral cells: in juxtaglomerular cells of tumoral tissue renin is synthesized as a preprorenin and rapidly converted into prorenin (55,000 mol wt), which is in turn packaged in secretory granules where it is processed into active renin (44,000 mol wt) and finally secreted; in the cultured tumoral cells renin is still biosynthesized as a preprorenin molecule and then converted into prorenin, but is neither stored as granules nor processed into active renin. In this case the renin is released in an inactive form.

Inhibition of renin: an updated review of the development of renin inhibitors.

As an important cascade involved in the regulation of blood pressure and volume homeostasis, the renin-angiotensin system (RAS) has been targeted for blood pressure control. In the last decades, the most successful strategy to control the RAS has been the inhibition of the angiotensin-converting enzyme (ACE) and the angiotensin type 1 (AT(1)) receptor. Small-molecule inhibitors targeting these steps have been widely used clinically. However, complete inhibition of the RAS is not achievable by blocking the ACE or the AT(1) because of the compensatory rise in plasma renin activity, which limits the efficacy of many RAS drugs. Direct inhibition of renin, the first and rate-limiting step in the RAS cascade, is the preferred strategy for controlling the RAS, and much progress has been made in the research and development of renin inhibitors. This review covers the evolution of renin inhibitors and discusses the advantages of renin inhibition at the enzymatic and biosynthetic levels for blood pressure control.

Purification of human plasma inactive renin by immunoaffinity chromatography on profragment-specific IgG.

Inactive renin was purified to apparent homogeneity from human plasma by ion exchange, gel filtration, Affi-Gel blue, immunoaffinity chromatography on profragment-specific IgG coupled to Sepharose, and preparative HPLC. By this method, a 460000-fold purification was obtained. The purified renin was totally inactive and was activated by trypsin.

Direct measurement of high molecular weight forms of renin in plasma.

Gel filtration of normal mouse serum revealed the presence of high molecular-weight forms of renin, in addition to the well-recognized 40 000 dalton form. The distribution of renin was measured by using both the antibody trapping method, which measures renin's enzymatic reactivity, and by a direct radio-immunoassay, which detects the renin molecule by its antigenic properties. A well characterized and distinct peak corresponding to a molecular weight of 200 000 or greater was always present. A less distinct peak was seen between 70 000 and 140 000 daltons. The well separated and symmetrical peak at 40 000-45 000 daltons was secreted from both kidneys and submaxillary glands and disappeared almost completely after combined nephrectomy and sialoadenectomy in contrast to the forms of higher molecular weight. The higher the molecular weight the lower was the specific enzymatic reactivity found to be. The 200 000-dalton peak was mainly detectable by the direct assay, although it was demonstrated to contain enzymatic reactivity to a small extent. The 40 000-dalton peak had a specific enzymatic reactivity and molecular weight identical to that of pure submaxillary renin.

A direct radioimmunoassay for plasma renin in mice and its evaluation.

The pioneering work of Cohen, S., Taylor J.M., Murakami, K., Michelakis, A.M. and Inagami, T. ((1972) Biochemistry 11 4286-4293) with isolation of submaxillary renin and the first direct radioimmunoassay for renin subsequently described by Michelakis A.M. Yoshida H., Menzie, J., Murakami, K. and Inagami, T ((1974) Endocrinology 94, 1101-1105) was reinvestigated and confirmed. In addition a detailed evaluation of the assay is described by characterization of the immunoreactivity of the labelled renin and demonstration of immunological identity between plasma renin and purified renin standards. Assay and equilibrium conditions were characterized and optimized and the association constant for the antibodies determined to be of the order of 10(11)1/mol. The validity of the direct assay for renin was further investigated by a comparison with another radioimmunoassay for renin; the antibody trapping method, which measures the enzymatic activity of renin in plasma. The correlation coefficient is high (0.91) but does not exclude that the direct assay measures a hypothetical prorenin in plasma as well.

High levels of active 40 000-dalton renin in mouse saliva, but no evidence of inactive or high molecular weight forms.

The presence of renin in parasympathetically elicited mouse saliva was demonstrated by using both the antibody trapping method, which measures renin's enzymatic activity, and a direct radioimmunoassay, which detects the renin molecule by its antigenic properties. Crossed immunoelectrophoresis of saliva samples using an antiserum elicited against pure submaxillary renin showed only one precipitation line, indicating the presence of only one form of renin. The position of the line was similar to that found when submaxillary gland extract was subjected to crossed immunoelectrophoresis. Tandem crossed immunoelectrophoresis showed complete identity between antigenic determinants in submaxillary and salivary renin. An apparent molecular weight of about 35 000 and 38 000 was found when saliva samples were subjected to gel filtration on Ultrogel AcA 44 and Sephadex G-100, respectively. No high molecular weight forms were present and no inactive forms could be demonstrated after limited pepsin or trypsin proteolysis. The specific enzymatic activity of renin in pilocarpine saliva was 0.37 Goldblatt Units (G.U.) . microgram-1, which is identical to that of pure submaxillary gland renin (0.41 G.U. . microgram-1) and to that to the storage form of renin in the submaxillary gland (0.4 G.U. . microgram-1) An identical Km value was found for salivary renin, 1.01 microM, and for pure submaxillary renin, 0.98 microM. It is concluded that renin in pilocarpine-elicited saliva is similar to the storage form of renin in the submaxillary gland with respect to molecular weight, enzymatic and immunological properties.

Probing epitopes on human prorenin during its proteolytic and non-proteolytic activation.

The conformational changes of prorenin (PR) that are associated with its reversible non-proteolytic activation and irreversible proteolytic activation were monitored with immunoradiometric assays, using antibodies against epitopes belonging to the propeptide or the renin part of PR. Binding of PR to the renin inhibitor remikiren or protonation of PR resulted in the slowly progressive and simultaneous expression (t1/2 congruent with3.5-5.0 h at 4 degreesC) of epitopes of the N-terminal and C-terminal halves of the propeptide and an epitope that is manifest on renin but not on native non-activated PR. During reversible PR activation-inactivation, expression and disappearance of these epitopes coincided with the appearance and disappearance of enzyme activity. Cleavage of the propeptide from the renin part of PR by plasmin, as demonstrated by the failure of remikiren to unmask the N-terminal and C-terminal propeptide epitopes, was, with some time lag, followed by the simultaneous expression (t1/2 congruent with60 min at 4 degreesC) of the renin-specific epitope and enzymatic activity. Based on these findings we propose a model for the non-proteolytic activation of PR that involves the formation of an intermediary form of activated PR with the following properties: (1) the covalently bound propeptide has moved out of the active-site cleft, so that binding sites are exposed to active site ligands, (2) the propeptide is still not in the 'relaxed' conformation that is characteristic for fully, non-proteolytically, activated PR, and (3) the N-terminal part of the renin polypeptide chain has not yet attained the proper location that is required for enzymatic activity.