Medication adherence during laboratory workup for primary aldosteronism: pilot study.

PURPOSE: Current hypertension guidelines stipulate that all incompatible medications be stopped before performing laboratory screening for aldosteronism, but patient adherence is unclear. We measured plasma drug concentrations to determine drug adherence and potential drug bias during biochemical tests. PATIENTS AND METHODS: Plasma concentrations of 10 antihypertensive drugs were quantified by mass spectrometry in 24 consecutive ambulatory patients with uncontrolled hypertension routinely evaluated for aldosteronism. Drug screening was done before (first visit), and on the day of biochemical tests (second visit) after stopping all incompatible medications. Concentrations above those expected at trough dosing interval defined same-day dose intake. RESULTS: On the first and second visits, 76% vs 77% of prescribed antihypertensive doses could be verified in plasma. A total of 33% of patients were found to be nonadherent and showed divergent plasma drug results relative to prescriptions (21% drugs not detected/13% unprescribed drugs found) on first visit, 25% on the second (0%/25%), and 46% for both. A total of 21% used medication incompatible with the biochemical tests on the second visit. Moreover, 17% of drug concentrations were below expected trough levels on the first vs 15% on the second visit. This analysis revealed additional four (17%) vs three (13%) nonadherent patients who failed same-day dose intake and remained undetected by qualitative drug tests. CONCLUSION: Nonadherence was frequent during laboratory evaluations for aldosteronism advocating cautious interpretation of results. A multicenter study is desirable to set the stage for new screening protocols that should incorporate also incentives and checks of drug adherence.

Angiotensinergic Innervation of the Human Right Atrium: Implications for Cardiac Reflexes.

BACKGROUND: The right atrium is densely innervated and provides sensory input to important cardiocirculatory reflexes controlling cardiac output and blood pressure. Its angiotensin (Ang) II-expressing innervation may release Ang II as a neuropeptide cotransmitter to modulate reflexes but has not yet been characterized. METHODS: Intraoperative surgical biopsies from human right atria (n = 7) were immunocytologically stained for Ang II, tyrosine hydroxylase (TH), and synaptophysin (SYN). Tissue angiotensins were extracted and quantified by radioimmunoassay. RESULTS: Angiotensinergic fibers were frequent in epicardial nerves and around vessels with variable TH co-localization (none to >50%/bundle). Fibers were also widely distributed between cardiomyocytes and in the endocardium where they were typically nonvaricose, TH/SYN-negative and usually accompanied by varicose catecholaminergic fibers. In the endocardium, some showed large varicosities and were partially TH or SYN-positive. A few endocardial regions showed scattered nonvaricose Ang fibers ending directly between endothelial cells. Occasional clusters of thin varicose terminals co-localizing SYN or TH were located underneath, or protruded into, the endothelium. Endocardial density of Ang and TH-positive fibers was 30-300 vs. 200-450/mm2. Atrial Ang II, III, and I concentrations were 67, 16, and 5 fmol/g (median) while Ang IV and V were mostly undetectable. CONCLUSIONS: The human right atrium harbors an abundant angiotensinergic innervation and a novel potential source of atrial Ang II. Most peripheral fibers were noncatecholaminergic afferents or preterminal vagal efferents and a minority was presumably sympathetic. Neuronal Ang II release from these fibers may modulate cardiac and circulatory reflexes independently from plasma and tissue Ang II sources.

Resetting of renal tissular renin-angiotensin and bradykinin-kallikrein systems after unilateral kidney denervation in rats.

The renal tissular renin-angiotensin and bradykinin-kallikrein systems control kidney function together with the renal sympathetic innervation but their interaction is still unclear. To further elucidate this relationship, we investigated these systems in rats 6 days after left kidney denervation (DNX, n = 8) compared to sham-operated controls (CTR, n = 8). Plasma renin concentration was unchanged in DNX vs. CTR (p = NS). Kidney bradykinin (BK) and angiotensin (Ang) I and II concentrations decreased bilaterally in DNX vs. CTR rats (~20 to 40%, p < 0.05) together with Ang IV and V concentrations that were extremely low (p = NS). Renin, Ang III and dopamine concentrations decreased by ~25 to 50% and norepinephrine concentrations by 99% in DNX kidneys (p < 0.05) but were unaltered in opposite kidneys. Ang II/I and KA were comparable in DNX, contralateral and CTR kidneys. Ang III/II increased in right vs. DNX or CTR kidneys (40-50%, p < 0.05). Ang II was mainly located in tubular epithelium by immunocytological staining and its cellular distribution was unaffected by DNX. Moreover, the angiotensinergic and catecholaminergic innervation of right kidneys was unchanged vs. CTR. We found an important dependency of tissular Ang and BK levels on the renal innervation that may contribute to the resetting of kidney function after DNX. The DNX-induced peptide changes were not readily explained by kidney KA, renin or plasma Ang I generation. However, tissular peptide metabolism and compartmentalization may have played a central role. The mechanisms behind the concentration changes remain unclear and deserve further clarification.

Pharmacotherapy: Optimal blockade of the renin-angiotensin-aldosterone system.

Inhibition of the renin­angiotensin­aldosterone system (RAAS) is a successful therapy for hypertension, heart failure, and renal insufficiency. Overdosing of RAAS inhibitors is occasionally observed when several agents are used together, and can cause adverse events such as hypotension, hyperkalaemia, and renal failure. We advocate optimal, rather than complete, RAAS blockade.

Angiotensinergic innervation of the kidney: present knowledge and its significance.

Intrarenal neurotransmission implies the co-release of neuropeptides at the neuro-effector junction with direct influence on parameters of kidney function. The presence of an angiotensin (Ang) II-containing phenotype in catecholaminergic postganglionic and sensory fibers of the kidney, based on immunocytological investigations, has only recently been reported. These angiotensinergic fibers display a distinct morphology and intrarenal distribution, suggesting anatomical and functional subspecialization linked to neuronal Ang II-expression. This review discusses the present knowledge concerning these fibers, and their significance for renal physiology and the pathogenesis of hypertension in light of established mechanisms. The data suggest a new role of Ang II as a co-transmitter stimulating renal target cells or modulating nerve traffic from or to the kidney. Neuronal Ang II is likely to be an independent source of intrarenal Ang II. Further physiological experimentation will have to explore the role of the angiotensinergic renal innervation and integrate it into existing concepts.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers.

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

Angiotensinergic neurotransmission in the peripheral autonomic nervous system.

Angiotensin (Ang) II has for long been identified as a neuropeptide located within neurons and pathways of the central nervous system involved in the control of thirst and cardio-vascular homeostasis. The presence of Ang II in ganglionic neurons of celiac, dorsal root, and trigeminal ganglia has only recently been described in humans and rats. Ang II-containing fibers were also found in the mesenteric artery and the heart, together with intrinsic Ang II-containing cardiac neurons. Ganglionic neurons express angiotensinogen and co-localize it with Ang II. Its intraneuronal production as a neuropeptide appears to involve angiotensinogen processing enzymes other than renin. Immunocytochemical and gene expression data suggest that neuronal Ang II acts as a neuromodulatory peptide and co-transmitter in the peripheral autonomic, and also sensory nervous system. Neuronal Ang II probably competes with humoral Ang II for effector cell activation. Its functional role, however, still remains to be determined. Angiotensinergic neurotransmission in the autonomic nervous system is a potential new target for therapeutic interventions in many common diseases such as essential hypertension, heart failure, and cardiac arrhythmia.

Blood pressure and renin-angiotensin system resetting in transgenic rats with elevated plasma Val5-angiotensinogen.

OBJECTIVE: Increases in plasma angiotensinogen (Ang-N) due to genetic polymorphisms or pharmacological stimuli like estrogen have been associated with a blood pressure (BP) rise, increased salt sensitivity and cardiovascular risk. The relationship between Ang-N, the resetting of the renin-angiotensin system, and BP still remains unclear. Angiotensin (Ang) II-induced genetic hypertension should respond to lisinopril treatment. METHODS: A new transgenic rat line (TGR) with hepatic overexpression of native (rat) Ang-N was established to study high plasma Ang-N. The transgene contained a mutation producing Val(5)-Ang-II, which was measured separately from nontransgenic Ile-Ang-II in plasma and renal tissue. RESULTS: Male homozygous TGR had increased plasma Ang-N (~20-fold), systolic BP (ΔBP+26 mmHg), renin activity (~2-fold), renin activity/concentration (5-fold), total Ang-II (~2-fold, kidney 1.7-fold) but decreased plasma renin concentrations (-46%, kidney -85%) and Ile(5)-Ang-I and II (-93%, -94%) vs. controls. Heterozygous TGR exhibited ~10-fold higher plasma Ang-N and 17 mmHg ΔBP. Lisinopril decreased their SBP (-23 vs. -13 mmHg in controls), kidney Ang-II/I (~3-fold vs. ~2-fold) and Ile(5)-Ang-II (-70 vs. -40%), and increased kidney renin and Ile(5)-Ang-I (>2.5-fold vs. <2.5-fold). Kidney Ang-II remained higher and renin lower in TGR compared with controls. CONCLUSION: High plasma Ang-N increases plasma and kidney Ang-II levels, and amplifies the plasma and renal Ang-II response to a given change in renal renin secretion. This enzyme-kinetic amplification dominates over the Ang-II mediated feedback reduction of renin secretion. High Ang-N levels thus facilitate hypertension via small increases of Ang II and may influence the effectiveness of renin-angiotensin system inhibitors.

Angiotensin II upregulates toll-like receptor 4 on mesangial cells.

Angiotensin II (AngII) mediates proinflammatory properties by activating NF-kappaB transcription factor nuclear translocation and inducing the expression of chemokines. For examination of whether AngII modulates the expression of Toll-like receptor 4 (TLR4), a key element of the innate immune system that senses LPS, mouse mesangial cells (MMC) were treated with AngII. AngII upregulated TLR4 mRNA and protein in MMC, and this effect was mediated through AngII type 1 receptors. Reporter gene experiments indicate that an activating protein-1 (AP-1) as well as an E-26 specific sequence (Ets) binding site in the TLR4 promoter are responsible for the AngII-stimulated transcriptional activity of the TLR4 gene. Preincubation of MMC with AngII enhanced LPS-induced NF-kappaB activation and chemokine expression. Immunohistochemical analyses revealed that double-transgenic rats that overexpressed human renin and angiotensinogen expressed higher levels of glomerular TLR4 compared with normal Sprague-Dawley rats. In vivo, infusion with AngII but not with norepinephrine into rats for 7 d also enhanced glomerular NF-kappaB activation after systemic application of LPS, suggesting that the effects are independent of concomitantly induced hypertension. Together, these observations suggest that AngII leads to an activation of the innate immune system by a novel mechanism involving the upregulation of TLR4. Our data contribute to a better understanding of how exogenous infections may trigger renal autoimmune processes, particularly in pathophysiologic situations with high renal AngII concentrations. Because TLR4 binds endogenous ligands (e.g., extracellular matrix components) in addition to microbial products, AngII-mediated upregulation of TLR4 also could be relevant for the development of inflammation in many noninfectious renal diseases.

Advanced glycation end products and the kidney.

Advanced glycation end products (AGEs) are a heterogeneous group of protein and lipids to which sugar residues are covalently bound. AGE formation is increased in situations with hyperglycemia (e.g., diabetes mellitus) and is also stimulated by oxidative stress, for example in uremia. It appears that activation of the renin-angiotensin system may contribute to AGE formation through various mechanisms. Although AGEs could nonspecifically bind to basement membranes and modify their properties, they also induce specific cellular responses including the release of profibrogenic and proinflammatory cytokines by interacting with the receptor for AGE (RAGE). However, additional receptors could bind AGEs, adding to the complexity of this system. The kidney is both: culprit and target of AGEs. A decrease in renal function increases circulating AGE concentrations by reduced clearance as well as increased formation. On the other hand, AGEs are involved in the structural changes of progressive nephropathies such as glomerulosclerosis, interstitial fibrosis, and tubular atrophy. These effects are most prominent in diabetic nephropathy, but they also contribute to renal pathophysiology in other nondiabetic renal diseases. Interference with AGE formation has therapeutic potential for preventing the progression of chronic renal diseases, as shown from data of animal experiments and, more recently, the first clinical trials.

Advanced glycation end products: a possible link to angiotensin in an animal model.

Advanced glycation end products (AGEs) have been associated with progressive vascular and renal damage in a variety of pathological conditions such as renal failure and diabetes mellitus. The formation of AGEs is generally attributed to increased oxidative and carbonyl stress or hyperglycemia. Activation of the cellular receptor of AGE (RAGE) leads to subsequent cellular activation and proinflammatory responses. Angiotensin (Ang) produces cellular oxidative stress and similarly promotes end organ damage via its type 1 receptor. We investigated the interrelation between these two systems in a new transgenic rat (TGR) model with Ang II-dependent hypertension and renal damage and in nontransgenic controls. TGR showed increased systolic blood pressure (approximately 210 mmHg), proteinuria, and increased renal collagen I mRNA expression compared with normotensive nontransgenic controls. Immunohistochemical staining of kidney sections showed colocalization for Nepsilon-carboxy(methyl)lysine, RAGE, and NF-kappaB in TGR glomeruli. These features were absent in nontransgenic controls. Our observations suggest a possible link between Ang II-dependent end-organ damage and the AGE/RAGE axis in vivo. TGRs provide an excellent model to study the interrelation between the renin-angiotensin system and the AGE/RAGE axis in promoting cardiovascular end-organ damage, which would otherwise not be possible in humans.

Blood pressure-independent ETA and AT1 receptor blocker effects on the coronaries of rats harboring human renin and angiotensinogen genes.

BACKGROUND: Blood pressure-independent (BP) effects of angiotensin (Ang) II and endothelin (ET) on coronaries (remodeling) in high renin hypertension are incompletely understood. METHODS: We studied the effects of subdepressor doses of Ang II receptor (AT1) blockade with losartan (10 mg/kg/day gavage) and endothelin A receptor (ETA) blockade with LU135252 (30 mg/kg/day) on the coronaries of rats harboring human renin and angiotensinogen genes (dTGR). Nontransgenic Sprague-Dawley rats were controls. The rats were treated between the ages of 6 and 10 weeks. Coronary cross-sectional area [CSA; 0.79 x (external diameter2 - internal diameter2)], cell proliferation, and infiltration of monocytes/macrophages were determined. RESULTS: Monotherapy did not lower BP while combination treatment did (p < 0.05). All treatments reduced mortality (p < 0.01). CSA was decreased by all treatments compared to vehicle, independent of blood pressure (p < 0.05). Extensive proliferation by PCNA staining and infiltration of ED-1-positive cells was diminished by both treatment and the combination. CONCLUSIONS: The data show that Ang II promotes coronary inflammation and remodeling, in part independent of blood pressure but dependent upon ET signaling. Combination treatment directed at both pathways may improve outcome, independent of blood pressure reduction.

Reduced hypertension-induced end-organ damage in mice lacking cardiac and renal angiotensinogen synthesis.

Hypertension-induced damage of kidney and heart is of major clinical relevance, but its pathophysiology is only partially understood. As there is considerable evidence for involvement of angiotensin II, we generated a new mouse model by breeding angiotensinogen (AOGEN) deficient mice with transgenic animals expressing the rat AOGEN gene only in brain and liver. This genetic manipulation overcame the hypotension of AOGEN-deficient mice and even caused hypertension indistinguishable in its extent from the parent transgenic mice with an intact endogenous AOGEN gene. In contrast to normal mice, however, crossbred animals lacked detectable expression of AOGEN in kidney and heart. As a consequence they showed markedly reduced cardiac hypertrophy and fibrosis. Furthermore, hypertension-induced alterations in kidney histology and function were less pronounced in crossbred mice than in equally hypertensive animals expressing AOGEN locally. The dysmorphogenesis observed in kidneys from AOGEN-deficient mice was absent in mice expressing this gene only in liver and brain. Our results support an important role of local AOGEN expression in hypertension-induced end-organ damage but not in the development of the kidney.

Decreased susceptibility of cardiac function to hypoxia-reoxygenation in renin-angiotensinogen transgenic rats.

We tested the hypothesis that the renin-angiotensin system (RAS) protects the contractile function of the myocardium against the damaging effect of hypoxia-reoxygenation. For this purpose, the contractility of isolated papillary muscles from wild-type (WT) rats and from rats expressing human renin and angiotensinogen as transgenes (TGR) was compared. After 15 min of hypoxia, peak force (PF) was decreased to 24 +/- 5% of the normoxic values in TGR (n = 10) and to 18 +/- 1% in WT rats (n = 12). PF and relaxation rates recovered completely in TGR but not in WT rats during 45 min of reoxygenation. Improved contractility of the papillary muscles from TGR during hypoxia-reoxygenation correlated with increased glutathione peroxidase activities and creatine kinase (CK)-MB and CK-BB isoenzyme levels. On the other hand, inhibition of the RAS with ramipril (1 mg/kg body wt for 3 wk) in WT animals resulted in deterioration of the contractile function of the papillary muscles during reoxygenation compared with untreated rats. These findings suggest that activation of the RAS protects contractile function of the cardiac muscle against hypoxia-reoxygenation, possibly through changes in CK isoenzymes and enhanced antioxidant capacity.

Rats transgenic for human renin and human angiotensinogen as a model for gestational hypertension.

Animal models of gestational hypertension are problematic. A novel mouse model was described earlier. The dams in that study were transgenic for human angiotensinogen and the sires for human renin; human renin was expressed in and produced by the placenta. This model was adapted to the rat, which has greater utility in terms of chronic instrumentation and physiologic measurements. Female rats transgenic for human angiotensinogen were mated with rats transgenic for human renin. Telemetry BP increased on day 5 of pregnancy from 110/80 mmHg to as high as 180/140 mmHg, while heart rate increased slightly. The renin transgene was expressed in the placenta, which resulted in increased human plasma renin concentration from 0 to 937 +/- 800 ng angiotensin I ml/h; the values returned to 0 after delivery. Female rats transgenic for human renin that were mated with male rats transgenic for human angiotensinogen in contrast exhibited a decrease in BP. In these rats, human angiotensinogen in plasma remained undetectable. Double transgenic offspring of these transgenic rats developed hypertension and end-organ damage, regardless of the source of the transgenes. The conclusion is that transgenic rats that bear human renin and angiotensinogen genes make an attractive model for gestational hypertension. The rat model will have greater utility than the mouse model.