p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage

We investigated whether or not p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). dTGR, with or without p38 inhibitor (BIRB796; 30 mg/kg per day in the diet), and nontransgenic Sprague-Dawley rats were studied in 2 protocols. In protocol 1 (week 7), systolic blood pressure of untreated dTGRs was 204+/-4 mm Hg, but partially reduced after BIRB796 treatment (166+/-7 mm Hg), whereas Sprague-Dawley rats were normotensive. The cardiac hypertrophy index was unchanged in untreated and BIRB796-treated dTGRs. The beta-myosin heavy chain expression of BIRB796-treated hearts was significantly lower in BIRB796 compared with dTGRs, indicating a delayed switch to the fetal isoform. BIRB796 treatment significantly reduced cardiac fibrosis, connective tissue growth factor, tumor necrosis factor-alpha, interleukin-6, and macrophage infiltration. Albuminuria was not reduced in BIRB796-treated dTGRs. Tubular and glomerular damage with tumor necrosis factor-alpha expression was unaltered, although serum creatinine and cystatin C were normalized. Renal macrophage infiltration, fibrosis, and vessel damage were reduced. In protocol 2 (week 8), we focused on mortality and arrhythmogenic electrical remodeling. Mortality of untreated dTGRs was 100% but was reduced to 10% in the BIRB796 group. Cardiac magnetic field mapping showed prolongation of depolarization and repolarization in untreated dTGRs compared with Sprague-Dawley rats with a partial reduction by BIRB796. Programmed electrical stimulation elicited ventricular tachycardias in 81% of untreated dTGRs but only in 48% of BIRB796-treated dTGRs. In conclusion, BIRB796 improved survival, target organ damage, and arrhythmogenic potential in angiotensin II-induced target organ damage.     

In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3x10(10) plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.     

Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy

Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.     

Exogenous L-arginine ameliorates angiotensin II-induced hypertension and renal damage in rats

Experiments were performed to determine whether exogenous L-arginine could ameliorate angiotensin II-induced hypertension and renal damage. Rats were instrumented with chronic indwelling femoral venous and arterial catheters for infusions of drugs and measurement of conscious arterial pressure. Arterial blood pressure significantly increased from 124+/-1 to 199+/-4 mm Hg, after 9 days of continuous infusion of angiotensin II (20 ng/kg per minute; IV; n=6 to 9). In contrast, the increase in arterial pressure after 9 days of angiotensin II infusion was significantly blunted by 45% (P=0.0003) in rats coadministered L-arginine (300 microg/kg per minute; IV; n=7 to 9). The glomerular injury index was significantly greater in rats administered angiotensin II in comparison with rats administered saline vehicle (P<0.001). Coinfusion of L-arginine significantly increased plasma nitrate/nitrite concentrations (P<0.001) and completely prevented angiotensin II-induced glomerular damage (P<0.001). Angiotensin II infusion alone and combined angiotensin II plus L-arginine infusion significantly increased urinary albumin excretion. Albuminuria in rats administered angiotensin II plus L-arginine is likely to be because of increased intraglomerular pressure. Our experiments demonstrate that L-arginine can blunt angiotensin II-induced hypertension and associated renal damage. This latter observation is most exciting because it indicates that increasing NO bioavailability, in addition to lowering arterial pressure, can greatly reduce hypertension-induced renal damage.     

Complement activation in angiotensin II-induced organ damage

We tested whether or not complement activation participates in angiotensin (Ang) II-induced vasculopathy. We used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the human renin inhibitor aliskiren. Sprague-Dawley (SD) rats were controls. DTGR had increased blood pressure at week 5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7. C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-alpha, C1q, C3, C3c, and C5b-9 expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after Ang II, TNF-alpha, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-alpha and CRP induced C3 mRNA slightly in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation and cell infiltration occurred before the onset of albuminuria in Ang II-mediated renal damage. TNF-alpha and CRP played a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang II-induced model, complement activation is a major participant and suggest that TNF-alpha and CRP may play a role in its induction.     

重组人松弛素体外调节血管紧张素Ⅱ诱导的大鼠心肌纤维化

目的:观察在重组人松弛素(rhRLX)及血管紧张素Ⅱ(AngⅡ)作用下对心脏成纤维细胞功能(CFs)的影响。方法:原代培养Sprague-Dawley大鼠心脏成纤维细胞,经AngⅡ(10-6mmol/L)单独作用及AngⅡ加rhRLX(100μg/L)共同作用后,用MTT比色法测定CFs的增殖;用ELISA法检测Ⅰ型前胶原羧基末端肽(PⅠCP)和Ⅲ型前胶原氨基末端肽(PⅢNP)的水平。结果:rhRLX可显著抑制AngⅡ促进CFs增殖,并拮抗AngⅡ促进CFs分泌PⅠCP的作用。结论:rhRLX能够改善AngⅡ诱导的心肌纤维化。     

NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy

Cardiac hypertrophy is an adaptive response to increases in blood pressure. Recent studies indicate that the hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. We hypothesize that superoxide anion mediates the hypertrophic response and that antioxidant therapy may be effective in attenuating cardiac hypertrophy. Neonatal rat cardiac myocytes were stimulated with angiotensin II (AngII, 1 microM) with and without various antioxidants. N-acetylcysteine (NAC, 10 mM) and probucol (50 microM), and to a lesser extent, vitamin C (500 microM) and reduced glutathione (1 mM), inhibited AngII-induced [(3)H]-leucine uptake and atrial natriuretic factor (ANF) promoter activity. The hypertrophic response is mediated by superoxide anion (O(2)(-).) since cell-permeable polyethylene glycol (PEG)-conjugated superoxide dismutase (50 U/ml), but not PEG-catalase (500 U/ml), attenuated AngII-induced [(3)H]-leucine uptake and ANF promoter activity. Furthermore, NAC blocked AngII-induced increase in myocardial oxidative stress, decreased the expression of ANF and myosin light chain-2v, and inhibited the re-organization of cytoskeletal proteins, desmin and alpha-actinin. These effects of AngII were abolished by angiotensin type 1 receptor blocker, losartan, but not type 2 receptor blocker, PD123319. Indeed, co-administration of losartan (10 mg/kg/d, 14 d) or NAC (200 mg/kg/d, 14 d) inhibited AngII-induced O(2)(-). production and cardiac hypertrophy in rats without affecting blood pressure. These findings indicate that the generation of O(2)(-). contributes to oxidant-induced hypertrophic response and suggest that antioxidant therapy may have beneficial effects in cardiac hypertrophy.     

Simvastatin prevents angiotensin II-induced cardiac alteration and oxidative stress

The influence of the HMG-CoA reductase inhibitor simvastatin was assessed on the cardiovascular alterations and production of free radicals associated with chronic angiotensin II (Ang II) infusion. Simvastatin (60 mg/kg per day PO) or placebo were given concomitantly for 10 days in Sprague-Dawley rats infused with Ang II (200 ng/kg per minute SC, osmotic pump). In addition, simvastatin or placebo was also given in vehicle-infused rats. Tail-cuff pressure and albuminuria were measured before and at the end of the treatment period. Cardiac weight, carotid structure, production of reactive oxygen species (ROS, by chemiluminescence) by polymorphonuclear leukocytes and aortic wall as well as protein and lipid oxidation products were determined at the end of the study. Ang II increased tail-cuff pressure by 56+/-12 mm Hg and simvastatin blunted the development of hypertension by approximately 70% (19+/-5 mm Hg). Increases in heart weight index and carotid cross-sectional area induced by Ang II were obliterated by simvastatin (3.18+/-0.09 versus 3.46+/-0.11 mg/g body wt and 0.125+/-0.010 versus 0.177+/-0.010 mm2, respectively). The Ang II-induced increases in leukocyte and aortic production of ROS as well as protein and lipid oxidation products were prevented by simvastatin. No effect of simvastatin was detected in non-Ang II-infused rats. These results indicate that simvastatin prevented the development of hypertension and cardiovascular hypertrophy together with inhibition of the induced angiotensin II production of ROS. Therefore, inhibition of HMG CoA reductase by statins may have a beneficial effect on cardiovascular alterations through its antioxidant action in experimental Ang II-dependent hypertension.     

Interleukin 6 underlies angiotensin II-induced hypertension and chronic renal damage

Chronic kidney disease (CKD) is a prevalent life-threatening disease frequently associated with hypertension, progression to renal fibrosis, and eventual renal failure. Although the pathogenesis of CKD remains largely unknown, an increased inflammatory response is known to be associated with the disease and has long been speculated to contribute to disease development. However, the causative factors, the exact role of the increased inflammatory cascade in CKD, and the underlying mechanisms for its progression remain unidentified. Here we report that interleukin 6 (IL-6) expression levels were significantly increased in the kidneys collected from CKD patients and further elevated in CKD patients characterized with hypertension. Functionally, we determined that angiotensin II is a causative factor responsible for IL-6 induction in the mouse kidney and that genetic deletion of IL-6 significantly reduced hypertension and key features of CKD, including renal injury and progression to renal fibrosis in angiotensin II-infused mice. Mechanistically, we provide both human and mouse evidence that IL-6 is a key cytokine functioning downstream of angiotensin II signaling to directly induce fibrotic gene expression and preproendothelin 1 mRNA expression in the kidney. Overall, both the mouse and human studies reported here provide evidence that angiotensin II induces IL-6 production in the kidney, and that, in addition to its role in hypertension, increased IL-6 may play an important pathogenic role in CKD by inducing fibrotic gene expression and ET-1 gene expression. These findings immediately suggest that the IL-6 signaling is a novel therapeutic target to manage this devastating disorder affecting millions worldwide.     

Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overexpression

OBJECTIVES AND DESIGN: Magnesium deficiency promotes vasoconstriction and myocardial damage. Recent studies provide evidence that Ang II mobilizes intracellular Mg through AT1 receptor-mediated pathways. We tested the hypothesis of whether magnesium supplementation prevents Ang II-induced myocardial damage and induction of the profibrotic connective tissue growth factor (CTGF). METHODS: Four-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGR) were given dietary magnesium supplementation (0.6%) for 3 weeks. Control dTGR and normotensive Sprague-Dawley (SD) rats received normal diet (Mg 0.2%). Histopathological, immunohistochemical and mRNA analysis were used to detect the treatment-related effects of dietary magnesium in dTGR. RESULTS: Magnesium (Mg) supplementation decreased blood pressure, ameliorated cardiac hypertrophy, protected against the development of Ang II-induced myocardial damage and increased serum ionized Mg2+ concentration (all variables P < 0.05). There was no difference in serum ionized Mg2+ concentration between dTGR and SD rats. Myocardial connective tissue growth factor (CTGF) mRNA and protein expressions were increased by 300% in dTGR (P < 0.05), especially in areas with myocardial infarctions and vascular inflammation. Magnesium supplementation prevented Ang II-induced myocardial CTGF overexpression (P < 0.05). Magnesium supplementation also improved the therapeutic effects of the calcineurin inhibitor tacrolimus, which produced marked hypomagnesemia when given as monotherapy. CONCLUSION: Our findings suggest a salutary effect for magnesium supplementation in the treatment of Ang II-induced myocardial complications.     

Role of osteopontin in cardiac fibrosis and remodeling in angiotensin II-induced cardiac hypertrophy

Osteopontin (OPN) is upregulated in several experimental models of cardiac fibrosis and remodeling. However, its direct effects remain unclear. We examined the hypothesis that OPN is important for the development of cardiac fibrosis and remodeling. Moreover, we examined whether the inhibitory effect of eplerenone (Ep), a novel aldosterone receptor antagonist, was mediated through the inhibition of OPN expression against cardiac fibrosis and remodeling. Wild-type (WT) and OPN-deficient mice were treated with angiotensin II (Ang II) for 4 weeks. WT mice receiving Ang II were divided into 2 groups: a control group and an Ep treatment group. Ang II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in WT mice. Ep treatment and OPN deficiency could reduce the Ang II-induced elevation of blood pressure and ameliorate the development of cardiac fibrosis, whereas Ep-only treatment abolished the development of cardiac hypertrophy. Most compelling, the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and subsequent left ventricular dilatation in Ang II-treated OPN-deficient mice. These results suggest that OPN has a pivotal role in the development of Ang II-induced cardiac fibrosis and remodeling. Moreover, the effect of Ep on the prevention of cardiac fibrosis, but not cardiac hypertrophy, might be partially mediated through the inhibition of OPN expression.     

Allopurinol attenuates oxidative stress and cardiac fibrosis in angiotensin II-induced cardiac diastolic dysfunction

Aims: Oxidative stress and fibrosis is implicated in cardiac remodeling and failure. We tested whether allopurinol could decrease myocardial oxidative stress and attenuate cardiac fibrosis and left ventricular diastolic dysfunction in angiotensin II (AngII)‐induced hypertensive mice. Methodology: We used 8‐week‐old male C57BL/6J mice, in which angiotensin II was subcutaneously infused for 4 weeks to mimic cardiac remodeling and fibrosis. They were treated with either normal saline or allopurinol in daily doses, which did not lower blood pressure. Results: Allopurinol improved diastolic dysfunction in angiotensin II‐induced hypertensive mice, which was associated with the amelioration of cardiac fibrosis. However, allopurinol showed no effect on the increased systolic blood pressure by angiotensin II infusion. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH/GSSG] was decreased and malondialdehyde levels were increased in the hearts of AngII‐treated mice. Allopurinol also inhibited both the decrease in the GSH/GSSG ratio and the increase in malondialdehyde levels in the heart. Infusion of AngII‐induced upregulation of transfer growth factor (TGF)‐β1, Smad3 expression and downregulation of Smad7 expression. Treatment with allopurinol reduced cardiac levels of TGF‐β1, Smad3, and increased Smad7 expression. Conclusions: These results suggest that allopurinol prevents pathological remodeling of the heart in AngII‐induced hypertensive mice. The antioxidative effect of allopurinol contributes to the regression of AngII‐induced cardiac diastolic dysfunction. These effects of allopurinol to prevent cardiac fibrosis are mediated at least partly through modulation of the TGF‐β1/Smad signaling pathway.     

Review of aldosterone- and angiotensin II-induced target organ damage and prevention

Aldosterone is well recognized as a cause of sodium reabsorption, water retention, and potassium and magnesium loss; however, it also produces a variety of other actions that lead to progressive target organ damage in the heart, vasculature, and kidneys. Aldosterone interacts with mineralocorticoid receptors to promote endothelial dysfunction, facilitate thrombosis, reduce vascular compliance, impair baroreceptor function, and cause myocardial and vascular fibrosis. Although angiotensin II has been considered the major mediator of cardiovascular damage, increasing evidence suggests that aldosterone may mediate and exacerbate the damaging effects of angiotensin II. While angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers reduce plasma aldosterone levels initially, aldosterone rebound, or 'escape' may occur during long-term therapy. Therefore, aldosterone blockade is required to reduce the risk of progressive target organ damage in patients with hypertension and heart failure. This may be achieved nonselectively with spironolactone or with use of the selective aldosterone blocker eplerenone. While both agents have been demonstrated to be effective antihypertensive agents, eplerenone may produce improved target organ protection as witnessed in a variety of clinical settings, without the antiandrogenic and progestational effects commonly observed with spironolactone.     

Pertussis toxin attenuates angiotensin II-induced vasoconstriction and inhibition of renin release

Angiotensin II can elicit cellular responses by 2 different receptor-dependent mechanisms: increase in intracellular calcium or inhibition of adenylate cyclase activity. The well-known inhibition of renin release from granulated cells of the kidney is thought to be mediated by an increase in intracellular calcium. However, the participation of the other possible pathway, i.e. inhibition of adenylate cyclase, has not been excluded. We studied this question by using the toxin from Bordetella pertussis, which inactivates the inhibitory coupling units Ni and thus permits to identify hormonal actions mediated through inhibition of adenylate cyclase. In isolated perfused kidneys from rats pretreated with pertussis toxin (2 micrograms/100 g i.v., single injection) the inhibition of renin release by angiotensin II (10(-11) to 10(-8) M) was significantly attenuated. In parallel, the vasoconstrictor response to angiotensin II was also diminished in these rat kidneys. The effect of pertussis toxin was apparent 3, 5 and 10 days after treatment, with a maximal effect at the fifth day. These data suggest that angiotensin II may exert the inhibitory effect on renin release in part through inhibition of adenylate cyclase in granulated cells of the kidney.     

P450-dependent arachidonic acid metabolism and angiotensin II-induced renal damage

Transgenic rats overexpressing both human renin and angiotensinogen genes (dTGR) develop hypertension, inflammation, and renal failure. We tested the hypothesis that these pathological features are associated with changes in renal P450-dependent arachidonic acid (AA) metabolism. Samples were prepared from 5- and 7-week-old dTGR and from normotensive Sprague-Dawley (SD) rats, ie, before and after the dTGR developed severe hypertension and albuminuria. At both stages, dTGR showed significantly lower renal microsomal AA epoxygenase and hydroxylase activities that reached 63% and 76% of the control values at week 7. Furthermore, the protein levels of several potential AA epoxygenases (CYP2C11, CYP2C23, and CYP2J) were significantly reduced. Immunoinhibition studies identified CYP2C23 as the major AA epoxygenase, both in dTGR and SD rats. Immunohistochemistry showed that CYP2C23 was localized in cortical and outer medullary tubules that progressively lost this enzyme from week 5 to week 7 in dTGR. CYP2C11 expression occurred only in the outer medullary tubules and was markedly reduced in dTGR compared with age-matched SD rats. These findings indicate site-specific decreases in the availability of AA epoxygenase products in the kidney of dTGR. In contrast to renal microsomes, liver microsomes of dTGR and SD rats showed no change in the expression and activity of AA epoxygenases and hydroxylases. We conclude that hypertension and end-organ damage in dTGR is associated with kidney-specific downregulation of P450-dependent AA metabolism. Because the products of AA epoxygenation have anti-inflammatory properties, this alteration may contribute to uncontrolled renal inflammation, which is a major cause of renal damage in dTGR.     

抑制鞘氨醇激酶-2活性加重血管紧张素II诱导的心肌细胞肥大

目的 本实验旨在明确鞘氨醇激酶（sphingosine kinase,SphK）2在血管紧张素（angiotensin,Ang）II诱导的心肌细胞肥大中的作用。方法 分离并体外培养SD乳鼠心肌细胞。给予AngII（10 μmol/L）处理24h诱导心肌细胞肥大。AngII刺激时分别给予溶媒或SphK2特异性抑制剂ABC294640（1 μmol/L）共处理。采用蛋白免疫印迹法检测心肌细胞SphK2蛋白表达。采用酶联免疫吸附试验（enzyme-linked immunosorbant assay,ELISA）检测心肌细胞细胞核1-磷酸鞘氨醇（sphingosine 1-phosphate,S1P）水平。采用结晶紫染色观察AngII诱导的心肌细胞肥大程度。采用实时定量聚合酶链式反应（real time-polymerase chain reaction,RT-PCR）检测心肌细胞肥大标志基因心房钠尿肽（atrial natriuretic peptide,ANP）、脑钠尿肽（brain natriuretic peptide,BNP）和β-肌球蛋白重链（β-myosin heavy chain,β-MHC）mRNA表达水平。结果 与对照组比较,AngII处理上调心肌细胞SphK2蛋白表达和S1P浓度（均P<0.05）,并增加心肌细胞横截面积与ANP、BNP和β-MHC mRNA表达水平（均P<0.05）。与溶媒组比较,ABC294640处理显著降低了心肌细胞核S1P浓度,并进一步增加了AngII处理后的心肌细胞横截面积和肥大标志基因mRNA表达水平（均P<0.05）。结论 抑制SphK2活性加重AngII诱导的心肌细胞肥大。SphK2有可能成为治疗病理性心肌肥大的新靶点。