BACKGROUND

In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] blocks activation of MAPKs by <em>angiotensin</em> II. We studied the effect of Ang-(<em>1</em>-<em>7</em>) on signalling responses in LLC-PK(<em>1</em>) cells in normal (5 mM) or high (25 mM) glucose.

METHODS

The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-<em>1</em> (SHP-<em>1</em>) activity was measured after immunoprecipitation, cell protein synthesis was determined by [(3)H]-leucine incorporation and transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>), fibronectin and collagen IV were assayed by immunoblots and/or ELISA.

RESULTS

High glucose stimulated p38 MAPK. This response was inhibited by Ang-(<em>1</em>-<em>7</em>) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-<em>7</em><em>7</em>9. Ang-(<em>1</em>-<em>7</em>) increased SHP-<em>1</em> activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(<em>1</em>-<em>7</em>) on high glucose-stimulated p38 MAPK. Ang-(<em>1</em>-<em>7</em>) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta<em>1</em>. Conversely, Ang-(<em>1</em>-<em>7</em>) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.

CONCLUSIONS

These data indicate that in proximal tubular cells, binding of Ang-(<em>1</em>-<em>7</em>) to the receptor Mas stimulates SHP-<em>1</em>, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(<em>1</em>-<em>7</em>) selectively inhibits glucose-stimulated protein synthesis and TGF-beta<em>1</em>. In diabetic nephropathy, Ang-(<em>1</em>-<em>7</em>) may partly counteract the profibrotic effects of high glucose.

The <em>angiotensin</em> I (AI) metabolite, A(<em>1</em>-<em>7</em>), elicited a concentration-dependent dilator response (ED50>> or = 2 microM) in porcine coronary artery rings which was markedly attenuated by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine, and abolished after removal of the endothelium. This effect of the heptapeptide was not mimicked by AII, AIII or A(3-8) at comparable concentrations. The A(<em>1</em>-<em>7</em>)-induced relaxation was not affected by AT<em>1</em> or AT2 receptor blockade or cyclo-oxygenase inhibition, but was attenuated by the B2 receptor antagonist, Hoe <em>1</em>40, and augmented by the <em>angiotensin</em>-converting enzyme (ACE) inhibitor, quinaprilat. These findings suggest that the relaxation to A(<em>1</em>-<em>7</em>) was mediated by the release of NO from the coronary endothelium through activation of an, as yet unidentified, AT receptor, the occupation of which also seems to stimulate the release of vasoactive kinins. Since A(<em>1</em>-<em>7</em>) accumulates during ACE inhibition, this mechanism may contribute to the coronary dilator effect of ACE inhibitors in vivo.

BACKGROUND

Statins are effective in prevention of end-organ damage; however, the benefits cannot be fully explained on the basis of cholesterol reduction. We used an angiotensin II (Ang II)-dependent model to test the hypothesis that cerivastatin prevents leukocyte adhesion and infiltration, induction of inducible nitric oxide synthase (iNOS), and ameliorates end-organ damage.

METHODS

We analyzed intracellular targets, such as mitogen-activated protein kinase and transcription factor (nuclear factor-kappaB and activator protein-1) activation. We used immunohistochemistry, immunocytochemistry, electrophoretic mobility shift assays, and enzyme-linked immunosorbent assay techniques. We treated rats transgenic for human renin and angiotensinogen (dTGR) chronically from week 4 to 7 with cerivastatin (0.5 mg/kg by gavage).

RESULTS

Untreated dTGR developed hypertension, cardiac hypertrophy, and renal damage, with a 100-fold increased albuminuria and focal cortical necrosis. dTGR mortality at the age of seven weeks was 45%. Immunohistochemistry showed increased iNOS expression in the endothelium and media of small vessels, infiltrating cells, afferent arterioles, and glomeruli of dTGR, which was greater in cortex than medulla. Phosphorylated extracellular signal regulated kinase (p-ERK) was increased in dTGR; nuclear factor-kappaB and activator protein-1 were both activated. Cerivastatin decreased systolic blood pressure compared with untreated dTGR (147 +/- 14 vs. 201 +/- 6 mm Hg, P < 0.001). Albuminuria was reduced by 60% (P = 0.001), and creatinine was lowered (0.45 +/- 0.01 vs. 0.68 +/- 0.05 mg/dL, P = 0. 003); however, cholesterol was not reduced. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression was diminished, while neutrophil and monocyte infiltration in the kidney was markedly reduced. ERK phosphorylation and transcription factor activation were reduced. In addition, in vitro incubation of vascular smooth muscle cells with cerivastatin (0.5 micromol/L) almost completely prevented the Ang II-induced ERK phosphorylation.

CONCLUSIONS

Cerivastatin reduced inflammation, cell proliferation, and iNOS induction, which led to a reduction in cellular damage. Our findings suggest that 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase inhibition ameliorates Ang II-induced end-organ damage. We suggest that these effects were independent of cholesterol.

Lessons learned from the characterization of the biological roles of Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (<em>angiotensin</em> II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin-<em>angiotensin</em> system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of <em>angiotensin</em>ogen through the characterization of the expression and functions of the dodecapeptide Ang-(<em>1</em>-<em>1</em>2) [<em>angiotensin</em>-(<em>1</em>-<em>1</em>2)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(<em>1</em>-<em>1</em>2) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(<em>1</em>-<em>1</em>2) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(<em>1</em>-<em>1</em>2) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(<em>1</em>-<em>1</em>2) expression in human left atrial appendages.

It is documented that chronic renal diseases are gender related. The protective role of <em>angiotensin</em> II receptor <em>1</em> (AT<em>1</em>) blocker losartan against cisplatin (CP)-induced nephrotoxicity was reported in males, but the role of gender is not well known. Six groups of Wistar rats were studied. Rats were divided into two groups of males and females to receive losartan for 9 days plus a single dose of CP (<em>7</em> mg/kg) at day 3. Two positive control groups of males and females received the same regimen, except that they received saline instead of losartan. The negative control groups received saline instead of CP at day 3 and also saline instead of losartan. The blood samples were obtained, and the kidneys underwent histopathological investigations. All the CP-treated animals lost weight, but losartan promoted weight loss in females (p < 0.05). Coadministration of losartan and CP in females, but not in males, significantly increased the serum levels of blood urea nitrogen and creatinine when compared with the negative and positive control groups (p < 0.05). No significant differences were observed in serum levels of total proteins, magnesium, and nitrite between the groups. Administration of CP increased the kidney tissue damage score (KTDS) and normalized kidney weight (p < 0.05). However, in the presence of AT<em>1</em> blockade, the KTDS (nonsignificantly) and normalized kidney weight (significantly, p < 0.05) increased in the CP-treated females. Such an observation was not seen in males. Losartan may prevent CP-induced nephrotoxicity in males, but it promotes the CP-induced damage in females, which may be related to the renin-<em>angiotensin</em> system receptors in the kidneys.

The renin-<em>angiotensin</em>-aldosterone system (RAAS) was initially thought to be fairly simple. However, this idea has been challenged following the development of RAAS blockers, including renin inhibitors, <em>angiotensin</em>-converting-enzyme (ACE) inhibitors, type <em>1</em> <em>angiotensin</em> II (AT(<em>1</em>))-receptor blockers and mineralocorticoid-receptor antagonists. Consequently, new RAAS components and pathways that might contribute to the effectiveness of these drugs and/or their adverse effects have been identified. For example, an increase in renin levels during RAAS blockade might result in harmful effects via stimulation of the prorenin receptor (PRR), and prorenin-the inactive precursor of renin-might gain enzymatic activity on PRR binding. The increase in <em>angiotensin</em> II levels that occurs during AT(<em>1</em>)-receptor blockade might result in beneficial effects via stimulation of type 2 <em>angiotensin</em> II receptors. Moreover, <em>angiotensin</em> <em>1</em>-<em>7</em> levels increase during ACE inhibition and AT(<em>1</em>)-receptor blockade, resulting in Mas receptor activation and the induction of cardioprotective and renoprotective effects, including stimulation of tissue repair by stem cells. Finally, a role of <em>angiotensin</em> II in sodium and potassium handling in the distal nephron has been identified. This finding is likely to have important implications for understanding the effects of RAAS inhibition on whole body sodium and potassium balance.

This study was designed to improve and validate methods for the accurate and consistent quantitation of <em>angiotensin</em> (ANG) I and II levels in rat kidney and to determine the effects on renal ANG I and II of changes in dietary sodium intake and ANG-converting enzyme (ACE) inhibition. Kidneys from pentobarbital-anesthetized rats were rapidly removed and homogenized in methanol before extraction and purification of ANG peptides by solid-phase extraction and high-performance liquid chromatography (HPLC). Recoveries of <em>1</em>25I-ANG I and II were greater than 80%. Reversed-phase HPLC of the partially purified methanol extract showed that greater than <em>7</em>5% of the ANG I- and greater than 82% of the ANG II-like immunoreactivity coeluted with ANG I and II, respectively. Dietary sodium deprivation (0.003 meq/g) and excess (<em>1</em>.34 meq/g) for <em>7</em> days significantly (P less than 0.0<em>1</em>) increased and decreased renal ANG I (296 +/- 30 and 82.6 +/- <em>1</em>5.8 vs. <em>1</em>6<em>1</em> +/- <em>1</em>8 fmol/g) and ANG II (2<em>1</em>6 +/- <em>1</em>6 and 45.6 +/- <em>1</em><em>1</em>.8 vs. 98 +/- <em>1</em>6 fmol/g) contents, respectively. Plasma ANG I and II levels showed similar changes. ACE activity was significantly upregulated by sodium deprivation in both kidney (44% increase) and plasma (30% increase). In rats fed normal chow, infusion of enalaprilat for <em>1</em> h abolished plasma ACE activity but decreased renal ACE activity by only 58%. ACE inhibition increased renal and plasma ANG I levels 2.8- and <em>1</em>2-fold, respectively, and decreased renal and plasma ANG II levels <em>7</em>5-<em>7</em>8%.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal sodium transport is increased by the <em>angiotensin</em> type <em>1</em> receptor (AT(<em>1</em>)R), which is counterregulated by dopamine via unknown mechanisms involving either the dopamine type <em>1</em> (D(<em>1</em>)R) or dopamine type 5 receptor (D(5)R) that belong to the D(<em>1</em>)-like receptor family of dopamine receptors. We hypothesize that the D(<em>1</em>)R and D(5)R differentially regulate AT(<em>1</em>)R protein expression and signaling, which may have important implications in the pathogenesis of essential hypertension. D(<em>1</em>)R and D(5)R share the same agonists and antagonists; therefore, the selective effects of either D(<em>1</em>)R or D(5)R stimulation on AT(<em>1</em>)R expression in human renal proximal tubule cells were determined using antisense oligonucleotides selective to either D(<em>1</em>)R or D(5)R. We also determined the role of receptor tyrosine kinase and the proteosome on the D(<em>1</em>)R/D(5)R-mediated effects on AT(<em>1</em>)R expression and internalization. In renal proximal tubule cells, D(5)R (not D(<em>1</em>)R) decreased AT(<em>1</em>)R expression (half-life: 0.4<em>7</em>+/-0.<em>1</em>8 hours) and AT(<em>1</em>)R-mediated extracellular signal-regulated kinase <em>1</em>/2 phosphorylation (232+/-<em>1</em>8.9 U with <em>angiotensin</em> II [<em>1</em>0(-<em>7</em>) mol/L] versus 8<em>1</em>+/-8.9 U with <em>angiotensin</em> II [<em>1</em>0(-<em>7</em>) mol/L] and fenoldopam [D(<em>1</em>)R/D(5)R agonist; <em>1</em>0(-6) mol/L; P<0.05; n=6). The fenoldopam-induced decrease in AT(<em>1</em>)R expression was reversed by 4-amino-5-(4-chlorophenyl)-<em>7</em>-(t-butyl) pyrazolo (3,4-d) pyrimidine (c-Src tyrosine-kinase inhibitor) and clasto-lactacystin beta-lactone (proteasome inhibitor), demonstrating that the fenoldopam-mediated decrease in total cell AT(<em>1</em>)R expression is a result of a c-Src- and proteasome-dependent process. D(5)R stimulation decreases AT(<em>1</em>)R expression and is c-Src and proteasome dependent. The discovery of differential regulation by D(<em>1</em>)R and D(5)R opens new avenues for the development of agonists selective to either receptor subtype as targeted antihypertensive agents that can decrease AT(<em>1</em>)R-mediated antinatriuresis.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is located in several tissues and is highly expressed in renal proximal tubules, where it degrades the vasoconstrictor <em>angiotensin</em> II (ANG II) to ANG-(<em>1</em>-<em>7</em>). Accumulating evidence supports protective roles of ACE2 in several disease states, including diabetic nephropathy. A disintegrin and metalloprotease (ADAM) <em>1</em><em>7</em> is involved in the shedding of several transmembrane proteins, including ACE2. Our previous studies showed increased renal ACE2, ADAM<em>1</em><em>7</em> expression, and urinary ACE2 in type 2 diabetic mice (Chodavarapu H, Grobe N, Somineni HK, Salem ES, Madhu M, Elased KM. PLoS One 8: e62833, 20<em>1</em>3). The aim of the present study was to determine the effect of insulin on ACE2 shedding and ADAM<em>1</em><em>7</em> in type <em>1</em> diabetic Akita mice. Results demonstrate increased renal ACE2 and ADAM<em>1</em><em>7</em> expression and increased urinary ACE2 fragments (≈<em>7</em>0 kDa) and albumin excretion in diabetic Akita mice. Immunostaining revealed colocalization of ACE2 with ADAM<em>1</em><em>7</em> in renal tubules. Renal proximal tubular cells treated with ADAM<em>1</em><em>7</em> inhibitor showed reduced ACE2 shedding into the media, confirming ADAM<em>1</em><em>7</em>-mediated shedding of ACE2. Treatment of Akita mice with insulin implants for 20 wk normalized hyperglycemia and decreased urinary ACE2 and albumin excretion. Insulin also normalized renal ACE2 and ADAM<em>1</em><em>7</em> but had no effect on tissue inhibitor of metalloproteinase 3 (TIMP3) protein expression. There was a positive linear correlation between urinary ACE2 and albuminuria, blood glucose, plasma creatinine, glucagon, and triglycerides. This is the first report showing an association between hyperglycemia, cardiovascular risk factors, and increased shedding of urinary ACE2 in diabetic Akita mice. Urinary ACE2 could be used as a biomarker for diabetic nephropathy and as an index of intrarenal ACE2 status.

In vivo studies were conducted in Na-replete anesthetized male Wistar rats with denervated kidneys. Intrarenal injections of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>) at>> <em>1</em> nmol/kg produced a shallow dose-dependent decrease in renal blood flow that was mediated by the AT<em>1</em>-type ANG II receptor. A constant intrarenal infusion of ANG-(<em>1</em>-<em>7</em>) at 0.<em>1</em> and <em>1</em> nmol.min-<em>1</em>.kg-<em>1</em> had minimal effects on renal blood flow and blood pressure and resulted in an elevated urinary excretion of Na and water compared with the time-control saline-infused group. To determine whether ANG-(<em>1</em>-<em>7</em>) may have a direct action on tubular epithelium to inhibit Na reabsorption, we examined the effect of ANG-(<em>1</em>-<em>7</em>) on transport-dependent O2 consumption (Qo2) in fresh suspensions of rat proximal tubules in vitro. ANG-(<em>1</em>-<em>7</em>) inhibited Qo2 in a concentration-dependent fashion with a threshold concentration of approximately <em>1</em>00 pM. Stimulating Na-K-adenosinetriphosphatase (Na-K-ATPase) activity with nystatin caused a leftward shift of the inhibitory concentration-response curve to ANG-(<em>1</em>-<em>7</em>). The 22% inhibition of Qo2 by <em>1</em> pM ANG-(<em>1</em>-<em>7</em>) was abolished by pretreatment with 5 mM ouabain (Na-K-ATPase inhibitor), unaltered by pretreatment with <em>1</em> microM PD-<em>1</em>233<em>1</em>9 (AT2 receptor antagonist), partially attenuated by <em>1</em> microM losartan (AT<em>1</em> receptor antagonist), and abolished by <em>1</em> microM [Sar<em>1</em>, Thr8]ANG II (nonselective ANG receptor antagonist). Together these findings indicate that ANG-(<em>1</em>-<em>7</em>) has biological activity in the kidney and, at nonvasoconstrictor doses, results in increased Na and water excretion in vivo. One site of action is the proximal tubule, where ANG-(<em>1</em>-<em>7</em>) can inhibit an ouabain-sensitive Na-K-ATPase exit step in cellular Na transport. This novel inhibitory action of ANG-(<em>1</em>-<em>7</em>) appears to be mediated by an AT<em>1</em> receptor (minor component) and a non-AT<em>1</em>, non-AT2 ANG receptor (major component).

In experimental renal failure, increased intramyocardial arteriolar wall thickness, reduced myocardial capillary density, and increased cardiac interstitium are found. The extent to which such alterations can be modified by therapeutic interventions has not been investigated to date. The purpose of this study was to examine the effects of Ramipril, Nifedipine and Moxonidine on these structural changes. Sham-operated and subtotally nephrectomized (SNX) 300-g male Sprague-Dawley rats (N = <em>7</em> to <em>1</em><em>1</em>) were left untreated (N = 9) or treated with Ramipril (0.5 mg/kg body wt per day; N = <em>7</em>), Nifedipine (30 mg/kg body wt per day; N = 9), or Moxonidine (<em>1</em>0 mg/kg body wt per day; N = 8) for 8 wk. After perfusion fixation, heart and aorta were examined by stereological techniques. Aortic wall thickness was significantly higher in SNX than in sham-operated control rats and was similarly lowered by all three interventions. In contrast, the wall thickness of intramyocardial arterioles was significantly higher in SNX; this was prevented by Ramipril and Nifedipine, but not by Moxonidine. Intramyocardial capillary length density (Lv) was significantly lower and interstitial volume density (Vv) significantly higher in untreated SNX. Reduction of capillary length density was completely prevented by Moxonidine and in part by Ramipril. The increase in cardiac interstitial volume density was completely prevented by Ramipril and was partially prevented by Moxonidine or Nifedipine treatment. The following conclusions can be drawn from the results: (<em>1</em>) all agents normalize aortic wall thickness, but only calcium channel blockers and <em>angiotensin</em>-converting enzyme (ACE) inhibitors prevent intramyocardial arteriolar wall thickening: (2) intramyocardial arteriolar wall thickening, capillary rarefaction, and expansion of the cardiac interstitium are seen in SNX even after lowering the blood pressure to subnormal levels; i.e., changes in systemic blood pressure cannot completely explain the altered vascular structure in renal failure; (3) the effects of Ramipril, Nifedipine, and Moxonidine on cardiovascular structures in experimental renal failure are not completely accounted for by their hemodynamic actions.

BACKGROUND

Although angiotensin II (Ang II) blockade is rapidly becoming standard antifibrotic therapy in renal diseases, current data suggest that Ang II blockade alone cannot stop fibrotic disease. New therapies, such as antibodies to transforming growth factor-beta (TGF-beta), or drug combinations will be required to further slow or halt disease progression. Here, using the anti-Thy1 model of glomerulonephritis, the maximally therapeutic dose of the TGF-beta neutralizing mouse monoclonal antibody (1D11) was determined and compared with the maximally effective dose of enalapril. Then, the effect of combining both treatments at maximal doses was determined.

METHODS

After disease induction with the anti-Thy1 antibody, OX-7, increasing doses of 1D11 were given intraperitoneally (IP) on days 1, 3, and 5. Enalapril was administered in drinking water from day 1. The fibrotic response was assessed at day 6.

RESULTS

1D11 dose-dependently reduced fibrosis, with the 0.5 and 5 mg/kg doses showing maximal therapeutic effects, reducing period-acid Schiff (PAS) staining by 56% and 45%, respectively. Fibronectin and collagen I staining was reduced by 32% to 36%, respectively. Glomerular mRNA and production of fibronectin, plasminogen activator inhibitor-1 (PAI-1), TGF-beta1, and p-Smad2 protein were also reduced. The maximal therapeutic effects of 1D11 and enalapril alone were very similar. However, combination therapy led to further reduction in disease. Notably, matrix deposition was reduced by 80%.

CONCLUSIONS

While 1D11 or enalapril at maximal doses reduce fibrosis equally, simultaneous blockade of Ang II and TGF-beta reduces fibrotic disease considerably more, offering hope that such drug combinations may confer a therapeutic advantage over angiotensin blockade alone.

Chronic administration of recombinant human relaxin (rhRLX) to conscious, normotensive rats (male and female) increases cardiac output (CO) and global arterial compliance (ACg) and reduces systemic vascular resistance (SVR) with no change in mean arterial pressure (MAP). Effects (magnitude and temporal pattern) of relaxin on systemic hemodynamics and arterial properties in hypertensive animal models are not known. Accordingly, the major goal of the present study was to determine the cardiovascular effects of rhRLX in hypertensive rats using 2 models: Long-Evans rats chronically administered <em>angiotensin</em> II (AII) and spontaneously hypertensive rats (SHR). CO and systemic arterial load, as quantified by SVR and ACg, were obtained using methods reported previously by us. In rats with AII-induced hypertension, acute rhRLX administration (up to 6 hours) significantly increased CO and ACg (24.9+/-3.9 and 34.3+/-<em>1</em>2.6% above baseline, respectively) and significantly decreased SVR (<em>1</em><em>7</em>.2+/-3.5%) without changing MAP. In contrast, acute rhRLX administration to SHR and normotensive rats for up to 6 hours failed to produce any significant changes in CO, ACg, SVR, or MAP. However, chronic rhRLX administration (<em>1</em> to <em>7</em> days) to SHR yielded significant changes (24.0+/-8.<em>1</em> and 22.3+/-6.6% increases in CO and ACg, respectively, and a <em>1</em>3.3+/-5.3% decrease in SVR, with no change in MAP). In conclusion, rhRLX increases CO and reduces arterial load in hypertensive rats without reducing MAP. However, the time course of response to rhRLX treatment is dependent on the model of hypertension such that rats characterized by AII-mediated hypertension responded more rapidly to rhRLX administration than SHR.

In this study we evaluated the effect of <em>angiotensin</em>(<em>1</em>-<em>7</em>) and its nonpeptide analog, AVE 099<em>1</em>, on the endothelial function in vivo. The experiments were performed in conscious adult male Wistar rats, with polyethylene catheters implanted into the descending aorta (through left carotid artery), for injection of acetylcholine or sodium nitroprusside, femoral artery for mean arterial pressure and heart rate measurement; and femoral vein for drug administration. Increasing doses of acetylcholine (3.<em>1</em> ng to 25.0 ng) or nitroprusside (<em>1</em>.0 microg to <em>1</em>0.0 microg) were administered before and 30 minutes after the start of the infusion of: <em>angiotensin</em>(<em>1</em>-<em>7</em>) (0.<em>7</em> and <em>7</em>.0 pmol/min); A-<em>7</em><em>7</em>9 (<em>1</em>80 pmol/min); <em>angiotensin</em>(<em>1</em>-<em>7</em>) (<em>7</em>.0 pmol/min) combined with A-<em>7</em><em>7</em>9 (<em>1</em>80 pmol/min); AVE 099<em>1</em> (<em>1</em><em>1</em>, 45, and 230 pmol/min); AVE 099<em>1</em> (45 pmol/min) combined with A-<em>7</em><em>7</em>9 (<em>1</em>80 pmol/min), or vehicle (6 microL/min). Baseline mean arterial pressure and heart rate were not altered during <em>angiotensin</em>(<em>1</em>-<em>7</em>) or AVE 099<em>1</em> infusion. <em>Angiotensin</em>(<em>1</em>-<em>7</em>) (0.<em>7</em> pmol/min) infusion produced a significant potentiation of the hypotensive effect of acetylcholine (3.<em>1</em> ng: -9+/-<em>1</em> mm Hg before; -<em>1</em>8+/-2 mm Hg after; P<0.05). A similar potentiation was observed with the higher dose of <em>angiotensin</em>(<em>1</em>-<em>7</em>). As observed for <em>angiotensin</em>(<em>1</em>-<em>7</em>), infusion of AVE 099<em>1</em> at 230 pmol/min potentiated the acetylcholine effect (3.<em>1</em> ng: -8+/-2 mm Hg before; -<em>1</em>6+/-2 mm Hg after; P<0.05). The potentiating effect was not observed for nitroprusside. A-<em>7</em><em>7</em>9 or l-NAME treatment blocked the potentiation produced by <em>angiotensin</em>(<em>1</em>-<em>7</em>) or AVE 099<em>1</em>. Our data indicate that short-term stimulation of <em>angiotensin</em>(<em>1</em>-<em>7</em>) receptors improve endothelial function through facilitation of nitric oxide release.

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] is a vasodilator peptide with cardiac and vascular protective properties. We examined the influence of Ang-(<em>1</em>-<em>7</em>), both endogenous and after chronic treatment with the peptide (5<em>7</em>6microg/(kgday)), on ischemia/reperfusion (I/R)-induced cardiac dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR). In isolated perfused hearts, recovery of left ventricular function from 40min of global ischemia was improved significantly in Ang-(<em>1</em>-<em>7</em>)- or captopril-treated diabetic SHR and worsened in animals treated with A<em>7</em><em>7</em>9, an Ang-(<em>1</em>-<em>7</em>) receptor (AT((<em>1</em>-<em>7</em>))) antagonist. The beneficial effect of captopril on cardiac recovery was reduced when co-administered with A<em>7</em><em>7</em>9. Cardiac NF-kappaB activity appears to be higher in diabetic SHR and treatment with Ang-(<em>1</em>-<em>7</em>) or captopril decreased NF-kappaB activity in diabetic SHR, an effect partially reversed by co-administration of A<em>7</em><em>7</em>9. Real-time PCR-based gene array analysis of cardiac tissue revealed that Ang-(<em>1</em>-<em>7</em>) or captopril treatment may reduce expression of several genes of inflammation involved in the NF-kappaB signalling pathway. The data provide for the first time a role for endogenous Ang-(<em>1</em>-<em>7</em>) as well as confirmation that exogenous treatment with the peptide produces cardioprotection. Whether potential anti-inflammatory and transcriptional factor changes are directly linked to the cardioprotection produced by Ang-(<em>1</em>-<em>7</em>) in diabetic SHR remains to be determined.

<em>1</em>. The hormonal control of glycogen breakdown was studied in hepatocytes isolated from livers of fed rats. 2. Glucose release was stimulated by [8-arginine]vasopressin (<em>1</em>0pm-<em>1</em>0nm), oxytocin (<em>1</em>nm-<em>1</em>mum), and <em>angiotensin</em> II (<em>1</em>nm-0.<em>1</em>mum). These responses are all at least as sensitive to hormone as is glucose output in the perfused rat liver. 3. The effect of these three hormones on glucose release was critically dependent on extracellular Ca(2+), unlike that of glucagon. Half-maximal restoration of the vasopressin response occurred if 0.3mm-Ca(2+) was added back to the incubation medium. 4. Glycogen breakdown was more than sufficient to account for the glucose released into the medium, in the absence or presence of hormones. Lactate release by hepatocytes was not affected by vasopressin, but was inhibited by glucagon. 5. If Ca(2+) was omitted from the extracellular medium, vasopressin stimulated glycogenolysis, but not glucose release. 6. The phosphorylase a content of hepatocytes was increased by vasopressin, oxytocin and <em>angiotensin</em> II; minimum effective concentrations were 0.<em>1</em>pm, 0.<em>1</em>nm and <em>1</em>0pm respectively. This response was also dependent on Ca(2+). <em>7</em>. These results demonstrate that hepatocytes can respond to low concentrations of vasopressin and <em>angiotensin</em> II, i.e. these effects are likely to be relevant in the intact animal. The role of extracellular Ca(2+) in the effects of these hormones on hepatic glycogenolysis and glucose release is discussed.

COVID-<em>1</em>9 is a rapidly spreading outbreak globally. Emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality. The SARS-CoV-2 infects humans through the <em>angiotensin</em> converting enzyme (ACE-2) receptor. The ACE-2 receptor is a part of the dual system renin-<em>angiotensin</em>-system (RAS) consisting of ACE-Ang-II-AT_<em>1</em>R axis and ACE-2-Ang-(<em>1</em>-<em>7</em>)-Mas axis. In metabolic disorders and with increased age, it is known that there is an upregulation of ACE-Ang-II-AT_<em>1</em>R axis with a downregulation of ACE-2-Ang-(<em>1</em>-<em>7</em>)-Mas axis. The activated ACE-Ang-II-AT<em>1</em>R axis leads to pro-inflammatory and pro-fibrotic effects in respiratory system, vascular dysfunction, myocardial fibrosis, nephropathy, and insulin secretory defects with increased insulin resistance. On the other hand, the ACE-2-Ang-(<em>1</em>-<em>7</em>)-Mas axis has anti-inflammatory and antifibrotic effects on the respiratory system and anti-inflammatory, antioxidative stress, and protective effects on vascular function, protects against myocardial fibrosis, nephropathy, pancreatitis, and insulin resistance. In effect, the balance between these two axes may determine the prognosis. The already strained ACE-2-Ang-(<em>1</em>-<em>7</em>)-Mas in metabolic disorders is further stressed due to the use of the ACE-2 by the virus for entry, which affects the prognosis in terms of respiratory compromise. Further evidence needs to be gathered on whether modulation of the renin <em>angiotensin</em> system would be advantageous due to upregulation of Mas activation or harmful due to the concomitant ACE-2 receptor upregulation in the acute management of COVID-<em>1</em>9.

The concentrations of <em>angiotensin</em> converting enzyme (ACE) activity, norepinephrine, and serotonin were measured in microdissected regions of the dog's brainstem and spinal cord. In addition, we determined the in vitro metabolism of <em>1</em>25I-<em>angiotensin</em> I (Ang I) in homogenates of the same brain punch regions. High ACE-specific activity was found in the monoamine-containing regions of the brainstem and in the intermediolateral column of the spinal cord. In brainstem homogenates <em>1</em>25I-Ang I was metabolized to <em>angiotensin</em> II (Ang-[<em>1</em>-8]) and the N-terminal heptapeptide Ang-(<em>1</em>-<em>7</em>). In the presence of MK 422 (50 microM), Ang-(<em>1</em>-<em>7</em>) was still generated, while the production of Ang-(<em>1</em>-8) was inhibited. This study revealed the presence of high ACE activity in monoamine regions of dog brainstem and spinal cord, and showed that the metabolite Ang-(<em>1</em>-<em>7</em>) is the major product generated from Ang I in the presence and absence of ACE inhibition.

Stimulation of the <em>angiotensin</em> II (Ang II) type <em>1</em> receptor (AT<em>1</em>-R) causes phosphorylation of extracellularly regulated kinases <em>1</em> and 2 (ERK<em>1</em>/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK<em>1</em>/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT<em>1</em>-R, and COS-<em>7</em> and human embryonic kidney (HEK) 293 cells transfected with the AT<em>1</em>-R. Ang II-induced ERK<em>1</em>/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-<em>7</em> cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK<em>1</em>/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM<em>1</em>9<em>7</em>, attenuated ERK<em>1</em>/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK<em>1</em>/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr3<em>1</em>9 residue of the AT<em>1</em>-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y3<em>1</em>9F) AT<em>1</em>-R expressed in COS-<em>7</em> cells caused EGF-R transactivation and subsequent ERK<em>1</em>/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.

The renin-<em>angiotensin</em> system, especially <em>angiotensin</em> II (ANG II), plays a key role in the development and progression of diabetic nephropathy. ANG <em>1</em>-<em>7</em> has counteracting effects on ANG II and is known to exert beneficial effects on diabetic nephropathy. We studied the mechanism of ANG <em>1</em>-<em>7</em>-induced beneficial effects on diabetic nephropathy in db/db mice. We administered ANG <em>1</em>-<em>7</em> (0.5 mg·kg(-<em>1</em>)·day(-<em>1</em>)) or saline to 5-mo-old db/db mice for 28 days via implanted micro-osmotic pumps. ANG <em>1</em>-<em>7</em> treatment reduced kidney weight and ameliorated mesangial expansion and increased urinary albumin excretion, characteristic features of diabetic nephropathy, in db/db mice. ANG <em>1</em>-<em>7</em> decreased renal fibrosis in db/db mice, which correlated with dephosphorylation of the signal transducer and activator of transcription 3 (STAT3) pathway. ANG <em>1</em>-<em>7</em> treatment also suppressed the production of reactive oxygen species via attenuation of NADPH oxidase activity and reduced inflammation in perirenal adipose tissue. Furthermore, ANG <em>1</em>-<em>7</em> treatment decreased lipid accumulation in db/db kidneys, accompanied by increased expressions of renal adipose triglyceride lipase (ATGL). Alterations in ATGL expression correlated with increased SIRT<em>1</em> expression and deacetylation of FOXO<em>1</em>. The upregulation of <em>angiotensin</em>-converting enzyme 2 levels in diabetic nephropathy was normalized by ANG <em>1</em>-<em>7</em>. ANG <em>1</em>-<em>7</em> treatment exerts renoprotective effects on diabetic nephropathy, associated with reduction of oxidative stress, inflammation, fibrosis, and lipotoxicity. ANG <em>1</em>-<em>7</em> can represent a promising therapy for diabetic nephropathy.

The JAK/STAT pathway is activated in vitro by <em>angiotensin</em> II (ANG II) and endothelin-<em>1</em> (ET-<em>1</em>), which are implicated in the development of diabetic complications. We hypothesized that ANG II and ET-<em>1</em> activate the JAK/STAT pathway in vivo to participate in the development of diabetic vascular complications. Using male Sprague-Dawley rats, we performed a time course study [days <em>7</em>, <em>1</em>4, and 28 after streptozotocin (STZ) injection] to determine changes in phosphorylation of JAK2, STAT<em>1</em>, and STAT3 in thoracic aorta using standard Western blot techniques. On day <em>7</em> there was no change in phosphorylation of JAK2, STAT<em>1</em>, and STAT3. Phosphorylation of JAK2, STAT<em>1</em>, and STAT3 was significantly increased on days <em>1</em>4 and 28 and was inhibited by treatment with candesartan (AT(<em>1</em>) receptor antagonist, <em>1</em>0 mg x kg(-<em>1</em>) x day(-<em>1</em>) orally in drinking water), atrasentan (ET(A) receptor antagonist, <em>1</em>0 mg x kg(-<em>1</em>) x day(-<em>1</em>) orally in drinking water), and AG-490 (JAK2 inhibitor, 5 mg x kg(-<em>1</em>) x day(-<em>1</em>) intraperitoneally). On day 28, treatment with all inhibitors prevented the significant increase in systolic blood pressure (SBP; tail cuff) of STZ-induced diabetic rats (SBP: <em>1</em>5<em>7</em> +/- 9.0, <em>1</em>30 +/- 3.3, <em>1</em>28 +/- 6.8, and <em>1</em>3<em>1</em> +/- <em>1</em>0.4 mmHg in STZ, STZ-candesartan, STZ-atrasentan, and STZ-AG-490 rats, respectively). In isolated tissue bath studies, diabetic rats displayed impaired endothelium-dependent relaxation in aorta (maximal relaxation: 95.3 +/- 3.0, 92.6 +/- <em>7</em>.4, <em>7</em>6.9 +/- <em>1</em>2.<em>1</em>, and 38.3 +/- <em>1</em>3.<em>1</em>% in sham, sham + AG-490, STZ + AG-490, and STZ rats, respectively). Treatment of rats with AG-490 restored endothelium-dependent relaxation in aorta from diabetic rats at <em>1</em>4 and 28 days of treatment. These results demonstrate that JAK2 activation in vivo participates in the development of vascular complications associated with STZ-induced diabetes.

BACKGROUND

Angiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte-endothelial cell interactions in vivo.

RESULTS

Intravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8+/-20. 7 versus 16.4+/-3.1 cells/min), adhesion (11.4+/-1.0 versus 0.8+/-0. 5 cells/100 microm), and emigration (4.0+/-0.7 versus 0.2+/-0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT(1) (losartan) or AT(2) (PD123,319) receptor antagonists significantly reduced Ang II-induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti-rat P-selectin monoclonal antibody abolished Ang II-induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation.

CONCLUSIONS

-Ang II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

BACKGROUND

The renin-<em>angiotensin</em> system (RAS) is activated in heart failure (HF) and inhibition of RAS is a mainstay therapy for HF. <em>Angiotensin</em>-converting enzyme 2 (ACE2) and its product, <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang-[<em>1</em>-<em>7</em>]), are important negative regulators of the RAS.

OBJECTIVE

A comprehensive examination of angiotensin peptide levels and therapeutic effects of recombinant human ACE2 (rhACE2) on peptide metabolism was evaluated in human plasma and explanted heart tissue from patients with HF.

METHODS

Using prospective cohorts with chronic (n = 59) and acute (n = 42) HF, plasma angiotensin analysis was performed using a unique liquid chromatography-mass spectrometry/mass spectroscopy method quantifying circulating and equilibrium levels. Angiotensin II (Ang II) metabolism was examined in human explanted hearts with dilated cardiomyopathy (n = 25).

RESULTS

The dynamic range of the RAS was large, with equilibrium <em>angiotensin</em> levels being 8- to <em>1</em>0-fold higher compared with circulating <em>angiotensin</em> levels. In chronic HF patients receiving ACE inhibition, plasma Ang II was suppressed and plasma Ang-(<em>1</em>-<em>7</em>) was elevated, whereas acute HF and patients receiving <em>angiotensin</em> receptor blocker had higher plasma Ang II with lower Ang-(<em>1</em>-<em>7</em>) levels. Suppressed Ang-(<em>1</em>-<em>7</em>)/Ang II ratio was associated with worsening HF symptoms and longer hospitalization. Recombinant human ACE2 effectively metabolized Ang-(<em>1</em>-<em>1</em>0) and Ang II into Ang-(<em>1</em>-9) and Ang-(<em>1</em>-<em>7</em>), respectively. Myocardial Ang II levels in explanted human hearts with dilated cardiomyopathy were elevated despite ACE inhibition with elevated chymase activity, and Ang II was effectively converted to Ang-(<em>1</em>-<em>7</em>) by rhACE2.

CONCLUSIONS

Plasma <em>angiotensin</em> peptides represent a dynamic network that is altered in HF and in response to rhACE2. An increased plasma Ang-(<em>1</em>-<em>7</em>) level is linked to ACE inhibitor use, whereas acute HF reduced Ang-(<em>1</em>-<em>7</em>) levels and suppressed the Ang-(<em>1</em>-<em>7</em>)/Ang II ratio. Increased chymase activity elevated Ang II levels in failing human hearts. Use of rhACE2 effectively normalized elevated Ang II while increasing Ang-(<em>1</em>-<em>7</em>) and Ang-(<em>1</em>-9) levels.

Obesity is characterized by a pro-inflammatory state commonly associated with type 2 diabetes and fat-liver disease. In the last few years, different studies pointed out the role of <em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) in the metabolic regulation. The aim of the present study was to evaluate the effect of oral-administration of Ang-(<em>1</em>-<em>7</em>) in metabolism and inflammatory state of high-fat feed rats. Twenty-four male Sprague Dawley rats were randomized into three groups: High Fat Diet (HFD); Standard Diet (ST); High Fat Diet+<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [HFD+Ang-(<em>1</em>-<em>7</em>)]. Glycemic profile was evaluated by glucose tolerance and insulin sensitivity tests, plasmatic glucose and insulin. Cholesterol, HDL and triglycerides analyses presented lipidic profile. RT-PCR evaluated mRNA expression to ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB genes. The main results showed that oral Ang-(<em>1</em>-<em>7</em>) decreased body weight and abdominal fat-mass. In addition, HFD+Ang-(<em>1</em>-<em>7</em>) treated rats presented enhanced glucose tolerance, insulin-sensitivity and decreased plasma-insulin levels, as well as a significant decrease in circulating lipid levels. These alterations were accompanied by a marked decreased expression of resistin, TLR4, ACE and increased ACE2 expression in liver. Furthermore, Ang-(<em>1</em>-<em>7</em>) decreases phosphorylation of MAPK and increases NF-κB expression. These alterations diminished expression of interleukin-6 and TNF-α, ameliorate inflammatory state in liver. In summary, the present study showed that oral-treatment with Ang-(<em>1</em>-<em>7</em>) in high-fat feed rats improved metabolism down-regulating resistin/TLR4/NF-κB-pathway.