Ang II-infused Rats Research Articles

Angiotensin II Infusion Induces Nephrin Expression Changes and Podocyte Apoptosis

Background/Aim: In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion. Methods: Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng·kg^–1·min^–1) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression. Results: The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = –0.63, p < 0.05). Conclusion: These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.

Purinergic receptors contribute to early mesangial cell transformation and renal vessel hypertrophy during angiotensin II-induced hypertension

Chronic ANG II infusions lead to increases in intrarenal ANG II levels, hypertension, and tissue injury. Increased blood pressure also elicits increases in renal interstitial fluid (RIF) ATP concentrations that stimulate cell proliferation. We evaluated the contribution of purinergic receptor activation to ANG II-induced renal injury in rats by treating with clopidogrel, a P2Y12 receptor blocker, or with PPADS, a nonselective P2 receptor blocker. alpha-Actin expression in mesangial cells, afferent arteriolar wall thickness (AAWT), cortical cell proliferation, and macrophage infiltration were used as early markers of renal injury. Clopidogrel and PPADS did not alter blood pressure, renin or kidney ANG II content. alpha-Actin expression increased from control of 0.6 +/- 0.4% of mesangial area to 6.3 +/- 1.9% in ANG II-infused rats and this response was prevented by clopidogrel (0.4 +/- 0.2%) and PPADS. The increase in AAWT from 4.7 +/- 0.1 to 6.0 +/- 0.1 mm in ANG II rats was also prevented by clopidogrel (4.8 +/- 0.1 mm) and PPADS. ANG II infusion led to interstitial macrophage infiltration (105 +/- 16 vs. 62 +/- 4 cell/mm(2)) and tubular proliferation (71 +/- 15 vs. 20 +/- 4 cell/mm(2)) and these effects were prevented by clopidogrel (52 +/- 4 and 36 +/- 3 cell/mm(2)) and PPADS. RIF ATP levels were higher in ANG II-infused rats than in control rats (11.8 +/- 1.9 vs. 5.6 +/- 0.6 nmol/l, P < 0.05). The results suggest that activation of vascular and glomerular purinergic P2 receptors may contribute to the mesangial cell transformation, renal inflammation, and vascular hypertrophy observed in ANG II-dependent hypertension.

Dose and time-dependent apoptotic effects by angiotensin II infusion on left ventricular cardiomyocytes

To gain insight into the regulation of cardiac apoptosis we studied the dose-response and time-course effects of angiotensin II (Ang II) infusion on ventricular cardiomyocyte apoptosis and on the expression of Bax and Bcl-2 genes and proteins. In the dose-response study, Ang II was infused subcutaneously at doses of 100, 200, 400, 800 and 1200 ng/kg per min for 14 days. In the time-course study, rats infused with Ang II at doses of 200 and 400 ng/kg per min were followed for 7 and 14 days. The cardiomyocyte apoptotic density was assessed by DNA end labelling (terminal deoxynucleotide nick-end labelling; TUNEL). Gene and protein expression of Bcl-2 and Bax were evaluated by reverse transcriptase-polymerase chain reaction and by Western blots. Systolic blood pressure and left ventricular mass were increased in a dose-dependent manner in Ang II-infused rats. A statistically significant increase in the rate of cardiac apoptosis and pro-apoptotic changes of Bcl-2 and Bax gene and protein expression was observed when high doses of Ang II (800-1200 ng/kg per min) were infused. A positive correlation of apoptotic density with Bax and a negative correlation with Bcl-2 and Bcl-2/Bax ratio were found. Cardiac apoptosis was greatly influenced by the timing of Ang II infusion. Losartan-treated Ang II-infused rats exhibited normalized systolic blood pressure, left ventricular weight, apoptosis, and Bax and Bcl-2 levels. Our results are consistent with the pathophysiological role of Ang II in induction of cardiac apoptosis, and explain the cardioprotective effect of Ang II receptor antagonists.

Crucial role of Rho-nuclear factor-κB axis in angiotensin II-induced renal injury

This study was performed to determine the effectiveness of the Rho kinase inhibitor and NF-kappaB inhibitor in renal injury of ANG II-infused hypertensive rats. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation (n = 7) or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG II-infused rats were further subdivided into three subgroups (n = 7 each) to receive one of the following treatments during the entire period: vehicle, Rho kinase inhibitor (fasudil; 3 mg.kg(-1).day(-1) ip), or NF-kappaB inhibitor (parthenolide; 1 mg.kg(-1).day(-1) ip). After 12 days of ANG II infusion, systolic blood pressure (BP; 208 +/- 7 vs. 136 +/- 3 mmHg), Rho kinase activity, NF-kappaB activity, renal ANG II contents (160 +/- 25 vs. 84 +/- 14 pg/g), monocytic chemotactic protein (MCP) 1 mRNA, interstitial macrophage infiltration, transforming growth factor-beta1 (TGF-beta1) mRNA, interstitial collagen-positive area, urinary protein excretion (43 +/- 6 vs. 11 +/- 2 mg/day), and urinary albumin excretion were significantly enhanced compared with the Sham group. While fasudil or parthenolide did not alter systolic BP (222 +/- and 190 +/- 21, respectively), both treatments completely blocked ANG II-induced enhancement of NF-kappaB activity, renal ANG II contents (103 +/- 11 and 116 +/- 21 pg/g, respectively), MCP1 mRNA, interstitial macrophage infiltration, TGF-beta1 mRNA, interstitial collagen-positive area, urinary protein excretion (28 +/- 6 and 23 +/- 3 mg/day, respectively), and urinary albumin excretion. Importantly, parthenolide did not alter ANG II-induced Rho kinase activation although fasudil abolished ANG II-induced Rho kinase activation. These data indicate that the Rho-NF-kappaB axis plays crucial roles in the development of ANG II-induced renal injury independently from BP regulation.

P22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats

Angiotensin II (ANG II) infusion increases renal superoxide (O(2)(-)) and enhances renal vasoconstriction via macula densa (MD) regulation of tubuloglomerular feedback, but the mechanism is unclear. We targeted the p22(phox) subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) with small-interfering RNA (siRNA) to reduce NADPH oxidase activity and blood pressure response to ANG II in rats. We compared single nephron glomerular filtration rate (SNGFR) in samples collected from the proximal tubule (PT), which interrupts delivery to the MD, and from the distal tubule (DT), which maintains delivery to the MD, to assess MD regulation of GFR. SNGFR was measured in control and ANG II-infused rats (200 ng.kg(-1).min(-1) for 7 days) 2 days after intravenous injection of vehicle or siRNA directed to p22(phox) to test the hypothesis that p22(phox) mediates MD regulation of SNGFR during ANG II. The regulation of SNGFR by MD, determined by PT SNGFR-DT SNGFR, was not altered by siRNA in control rats (control + vehicle, 13 +/- 1, n = 8; control + siRNA, 12 +/- 2 nl/min, n = 8; not significant) but was reduced by siRNA in ANG II-treated rats (ANG II + vehicle, 13 +/- 2, n = 7; ANG II + siRNA, 7 +/- 1 nl/min, n = 8; P < 0.05). We conclude that p22(phox) and NADPH oxidase regulate the SNGFR during ANG II infusion via MD-dependent mechanisms.

Interaction between Ang II and GABA systems in NTS: Central resetting of blood pressure regulation

There is little information concerning the interactions between Ang II and GABA systems in the NTS. Our previous studies, using gene microarrays, demonstrated that the GABA-B receptor (GBR) expression was increased following treatment of cultured NTS neurons with Ang II. In the present study, we examined the effects of Ang II on GBR expression in the NTS of SD rats. Ang II or saline control was infused subcutaneously for 2 weeks with osmotic minipumps. Ang II infusion induced a sustained increase in mean BP. GBR mRNA levels in NTS brain tissue were detected with real-time PCR showing GBR1 mRNA levels in the NTS were significantly increased in Ang II-infused rats. To rule out the possibility that the alterations of GBR expression observed in the above experiment is a result of the elevated BP, we examined the effect of Ang II on GBR expression in neurons cultured from NTS. Ang II treatment (100 nM, 5 hrs) induced a 2-fold increase in GBR mRNA levels. In addition, Superfusion of baclofen decreased the neuronal firing rate by 39% and 63% respectively in control and Ang II-treated neurons. In summary, these experiments indicate that Ang II stimulates GBR expression, consequently enhances GABAergic neurotransmission in the NTS. This enhanced GABAergic neurotransmission may lead to dampening of baroreflexes and consequently central resetting of long-term blood pressure regulation to a higher level in Ang II-related hypertension. Supported by AHA grant 0635050N

Synergistic actions of enalapril and tempol during chronic angiotensin II-induced hypertension

Experiments were designed to test the hypothesis that antioxidant treatment would increase the anti-hypertensive actions of endogenous kinins during angiotensin converting enzyme (ACE) inhibition. Four groups of rats, all given angiotensin II (Ang II) for 2 weeks, were studied: 1) control, 2) enalapril, 3) tempol or 4) both tempol and enalapril. Ang II significantly increased systolic blood pressure (BP) when compared with the baseline (170 ± 8 vs. 128 ± 4 mm Hg, P < 0.05). Neither enalapril nor tempol alone was able to attenuate the elevation in BP (165 ± 7 and 164 ± 6 mm Hg, respectively). In contrast, combined administration of tempol and enalapril prevented the increase in BP (137 ± 5 mm Hg). Plasma 8-isoprostane increased in Ang II-infused rats when compared with control untreated rats (69 ± 14 vs. 23 ± 0.5 pg/ml, P < 0.05). Tempol alone or tempol plus enalapril significantly attenuated the increase in plasma 8-isoprostane (29 ± 6 and 34 ± 7 pg/ml, respectively). In additional experiments, we used the bradykinin B 2 antagonist, icatibant to determine if increased B 2 receptor contributes to the anti-hypertensive effect of combined tempol and enalapril in Ang II-infused rats. Icatibant decreased the ability of this combination to lower arterial pressure. Additionally, a significant increase in B 1 receptor protein expression in renal cortex of Ang II-infused rats was observed compared to control suggesting that bradykinin receptor activation could account for the effect of enalapril to enhance the actions of tempol. These data support the hypothesis that combined reduction of superoxide along with enhanced endogenous kinins may facilitate blood pressure lowering in Ang II hypertension.

Rosiglitazone inhibits angiotensin II-induced CTGF expression in vascular smooth muscle cells––Role of PPAR-γ in vascular fibrosis

Angiotensin (Ang) II plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. Connective tissue growth factor (CTGF) is a potent profibrotic factor implicated in the Ang II-induced pathologic fibrosis process. PPAR-γ activators thiazolidinediones have been recently reported to have beneficial vascular effects. However, their effects and related molecular mechanisms on extracellular matrix (ECM) turnover in vascular smooth muscle cells (VSMCs) are unknown. The present study evaluated the regulation of Ang II-induced CTGF, ECM production and cell growth by rosiglitazone in VSMCs. In aorta of Ang II-infused rats, CTGF expression was markedly increased, and type III collagen and fibronectin overexpression was observed. Cotreatment with rosiglitazone diminished these changes, whereas increased nuclear PPAR-γ expression in VSMCs. In growth-arrested VSMCs, rosiglitazone attenuated the proliferation and apoptosis, increased PPAR-γ production and activation, and reduced CTGF and ECM production in response to Ang II in a dose-dependent fashion. These inhibitory effects were attenuated by the pretreatment of cells with PPAR-γ antagonist GW9662 or bisphenol A diglycidyl ether (BADGE). Furthermore, rosiglitazone inhibited Ang II-induced Smad2 production and phosphorylation but had no effect on transforming growth factor-β1 (TGF-β1) expression. These results suggest that in Ang II-stimulated VSMCs, rosiglitazone caused an antiproliferative, antiapototic effect and reduces ECM production through mechanisms that include reducing CTGF expression, and a crosstalk between PPAR-γ and Smad may be involved in the inhibitory effects of rosiglitazone. This novel finding suggests a role of PPAR-γ activators in preventing Ang II-induced vascular fibrosis.

Detection of specific nitrotyrosine-modified proteins as a marker of oxidative stress in cardiovascular disease

the oxidative modification of proteins is a natural consequence of aerobic life and is also recognized to play a crucial role in the pathological response of cells to increased oxidative stress. Several oxidative modifications of a protein can occur as a result of oxidative stress. These can range

Effects of Anesthesia on Plasma and Kidney ANG II Levels in Normotensive and ANG II-Dependent Hypertensive Rats

Background: Previous studies have implicated that normotensive rats with normal renal renin activity respond to anesthesia and surgery with greater increases in plasma and kidney angiotensin II (ANG II) concentrations than ANG II-dependent hypertensive rats with intrarenal renin depletion. In the present study, we therefore compared plasma and kidney ANG II levels in anesthetized and conscious normotensive and ANG II-dependent hypertensive rats. Methods: Salt-replete Hannover-Sprague-Dawley rats (HanSD) served as controls. As models of ANG II-dependent hypertension we used: 1st, transgenic rats harboring the Ren-2 renin gene (TGR); 2nd, two-kidney, one-clip (2K1C) Goldblatt hypertensive rats, and, 3rd, ANG II-infused hypertensive rats. As additional model with enhanced renin-angiotensin system (RAS) activity, salt-depleted HanSD and TGR were employed. Results: In anesthetized salt-repleted HanSD, plasma and kidney ANG II levels were higher than in salt-repleted TGR, ANG II-infused and 2K1C rats. Salt depletion caused marked increases in ANG II levels in HanSD but did not alter them in TGR. In contrast, in conscious animals immediately after decapitation plasma and kidney ANG II levels were similar in salt-repleted and salt-depleted TGR, in ANG II-infused rats, in the clipped kidney of 2K1C rats and in salt-depleted HanSD and in all these groups they were significantly higher than in salt-repleted HanSD. Conclusions: These findings indicate that anesthesia increases plasma and kidney ANG II levels in HanSD to a greater degree than in ANG II-dependent models of hypertension. Therefore, the results from studies employing anesthetized animals must be interpreted with caution.

The Rho-kinase pathway regulates angiotensin II-induced renal damage

Angiotensin II (AngII) is a key factor in the pathogenesis of renal damage. AngII via AngII type 1 receptors activates several intracellular signaling systems, including the small guanosine triphosphatase Rho and its downstream effector Rho-dependent serine-threonine kinase (Rho-kinase). The Rho/Rho-kinase pathway contributes to inflammatory and proliferative changes observed in cardiovascular diseases. However, the data on renal diseases are scarce. The aim of this study was to investigate the effect of Rho-kinase inhibition in AngII-induced renal damage. We used the model of systemic AngII infusion into normal rats (100 ng/kg per minute; subcutaneous osmotic minipumps), and some animals were treated with the Rho-kinase inhibitor Y-27632 (30 mg/kg per day). In the kidneys of these animals, we evaluated renal lesions, transcription factor activity (by electrophoretic mobility shift assay), and messenger RNA (by polymerase chain reaction) and protein expression levels (by Western blot and/or immunohistochemistry) of proinflammatory and profibrotic factors. Rats infused with AngII for three days present renal inflammatory cell infiltration and slight tubular damage, which were diminished by treatment with the Rho-kinase inhibitor Y-27632. AngII activates nuclear factor-kappaB and causes overexpression of proinflammatory factors, including cytokines (tumor necrosis factor alpha) and chemokines (monocyte chemotactic protein-1), and of profibrotic factors (connective tissue growth factor). Treatment of AngII-infused rats with Y-27632 decreases the upregulation of these proinflammatory and profibrotic mediators. These data demonstrate that the Rho-kinase pathway is involved in renal damage caused by AngII through the regulation of proinflammatory and profibrotic mediators. These results suggest that inhibition of the Rho-kinase pathway represents a novel therapy for renal diseases associated with local AngII generation.

Role of aldosterone in angiotensin II-induced cardiac and aortic inflammation, fibrosis, and hypertrophy

Activation of the renin-angiotensin-aldosterone system is associated with increased extracellular matrix and inflammatory markers in the cardiovascular system. We evaluated the effects of aldosterone antagonism on cardiovascular structure, collagen deposition, and expression of inflammatory markers in 2-week angiotensin (Ang) II-infused rats (120 ng.kg-1.min-1, s.c.)+/-spironolactone or hydralazine (25 mg.kg-1.d-1). Aortic and cardiac collagen density was evaluated with Sirius red staining. NFkappaB and AP-1 were measured by a electrophoretic mobility shift assay, and ED-1 (macrophage marker) and vascular cell adhesion molecule-1 (VCAM-1) were measured by immunohistochemistry. Ang II increased blood pressure (176+/-2 mmHg vs. 115+/-1 mmHg in controls, p<0.01), which was attenuated by spironolactone (147+/-4 mmHg, p<0.01) and prevented by hydralazine (124+/-2 mmHg, p<0.01). Ang II enhanced left ventricular interstitial collagen type I/III deposition (4.1%+/-0.1% vs. 3.1%+/-0.2%, p<0.05), and this was attenuated by spironolactone but not hydralazine. Ang II-induced cardiac perivascular fibrosis was prevented by spironolactone and hydralazine. Ang II significantly increased cardiac AP-1 activity and ED-1 expression, which was prevented by spironolactone only. Ang II-enhanced NFkappaB activity, and VCAM-1 expression was reduced by spironolactone and hydralazine, whereas aortic hypertrophy was prevented by spironolactone and slightly reduced by hydralazine. In conclusion, blockade of mineralocorticoid receptors with spironolactone inhibited Ang II-induced aortic hypertrophy, cardiac transcription factor activation, upregulation of downstream inflammatory markers, and collagen deposition, thus preventing Ang II-induced cardiovascular damage.

Angiotensin II Exerts Positive Feedback on the Intrarenal Renin-Angiotensin System by an Angiotensin Converting Enzyme-Dependent Mechanism 1

Plasma angiotensin II (ANG II) is not increased significantly in renovascular hypertension (RVH), but tissue ANG II levels are elevated in both kidneys of renovascular rats. Because the contralateral, non-ischemic kidney is critical for maintenance of hypertension in RVH, this study sought to understand the mechanism by which intrarenal ANG II levels are augmented in the non-ischemic kidney. This study tested the hypothesis that an incremental increase in plasma ANG II induces the intrarenal renin-angiotensin system (RAS) in the non-ischemic kidney by an angiotensin converting enzyme (ACE) dependent mechanism. To simulate the incremental increase in plasma ANG II induced by the ischemic kidney in RVH, an ANG II infusion model was used. This model used a chronic infusion of ANG II (40 ng/min) or vehicle by osmotic minipump into uninephrectomized rats. Parallel groups were treated with the ACE inhibitor Enalaprilat (200 mg/kg/day). Intrarenal ACE activity was measured by radioenzymatic assay. ANG II levels were quantified by radioimmunoassay. Hypertension was evident in ANG II-infused rats, compared to control rats (155 +/- 4 versus 112 +/- 1 mmHg; P < 0.001). Concurrent treatment with Enalaprilat reversed the hypertension induced by ANG II infusion (98 +/- 3 versus 155 +/- 4 mmHg; P < 0.001). ANG II up-regulated intrarenal ACE activity in the non-ischemic kidney (59.2 +/- 11.9 versus 25.2 +/- 6.8 units/mg protein; P < 0.01). Enalaprilat significantly decreased renal ACE activity in ANG II-treated rats, compared to ANG II alone (11.4 +/- 1.0 versus 59.2 +/- 11.9 units/mg protein; P < 0.001). Intrarenal ANG II was increased in ANG II-infused rats, compared to control animals (52.9 +/- 7.1 versus 23.0 +/- 3.2 fmol/mg tissue; P < 0.001), and Enalaprilat prevented ANG II-induced increases in intrarenal ANG II (29.9 +/- 2.6 versus 52.9 +/- 7.1 fmol/mg tissue; P < 0.05). Incremental changes in plasma ANG II induce de novo production of ANG II in the non-ischemic kidney to augment intrarenal ANG II content. ACE inhibition blocks this positive feedback loop, suggesting that ANG II activates the intrarenal RAS by an ACE-dependent mechanism. The impact of ACE inhibition on blood pressure suggests that this feedback loop may be an important mechanism for maintenance of hypertension in RVH.

Angiotensin Converting Enzyme-Independent Angiotensin II Production by Chymase is Up-regulated in the Ischemic Kidney in Renovascular Hypertension

Tissue angiotensin II (ANG II) levels are elevated in both kidneys in renovascular hypertension (RVH). It has been demonstrated previously that intrarenal ANG II is augmented by an angiotensin converting enzyme (ACE) dependent mechanism in the non-ischemic kidney, but the role of ACE-independent production of ANG II in the kidney by the enzyme chymase is unknown. This study tested the hypothesis that intrarenal chymase activity is up-regulated in RVH. A two-kidney, one-clip (2K1C) rat model was used to induce RVH (n = 6 rats/group). Regulation of intrarenal chymase activity by plasma ANG II was investigated using an ANG II-infusion model. At sacrifice 14 days post-operatively, steady-state ANG II levels in plasma and kidney were quantified by radioimmunoassay. ANG II production was quantified in kidney homogenates by incubating at 37 degrees C for 60 min with enzyme substrate (200 microm ANG I) alone or substrate containing the chymase inhibitor chymostatin. ANG II was separated and quantitated by HPLC. Chymase activity was defined as the fraction of ANG II production inhibited by Chymostatin. 2K1C and ANG II-infused rats developed significant hypertension, compared to control rats (P = 0.0001 and P = 0.001, respectively). Chymase-dependent ANG II production was increased in the ischemic kidney, but not the non-ischemic kidney, of 2K1C rats compared to control animals (*P < 0.05). Intrarenal chymase activity was unchanged by ANG II infusion (P = NS). Chymase activity is up-regulated in the ischemic kidney of 2K1C rats. Plasma ANG II does not appear to regulate intrarenal chymase activity, suggesting that ischemia per se up-regulates chymase activity in the kidney. ACE-independent ANG II production by chymase may provide a mechanism for augmenting intrarenal ANG II in the ischemic kidney in RVH.

AT1receptor-mediated enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats

Angiotensin II (ANG II)-infused rats exhibit increases in distal nephron renin expressed in principal cells of connecting tubules and collecting ducts. This study was performed to determine whether the augmentation of distal nephron renin involves ANG II type 1 (AT1) receptor activation. Male Sprague-Dawley rats (200-220 g) were divided into three groups: 1) sham operated (n = 8); 2) ANG II infused (80 ng/min, 13 days, n = 8); and 3) ANG II infused plus AT1 receptor blocker (ARB), olmesartan (5 mg/days, n = 8). ANG II infusion increased systolic blood pressure (BP; 178 +/- 4 vs. 122 +/- 1 mmHg; P < 0.001) and suppressed plasma renin activity (PRA; 0.08 +/- 0.1 vs. 5.3 +/- 0.8 ng ANG I x ml(-1) x h(-1)). ARB treatment prevented the increase in BP (113 +/- 6 mmHg) and led to increases in PRA (15.8 +/- 1.5 ng ANG I x ml(-1) x h(-1)). Renin protein levels measured in the kidney medulla, to avoid contribution from juxtaglomerular apparatus cells, were higher in ANG II-infused rats [1.64 +/- 0.3 vs. 1.00 +/- 0.1 densitometric units (DU) compared with sham-operated rats; P < 0.05], and ARB treatment prevented this increase (1.01 +/- 0.1). Similarly, renin immunoreactivity increased in medullary collecting ducts of ANG II-infused compared with sham-operated rats (2.5 +/- 0.3 vs. 1.0 +/- 0.2 DU; P < 0.001), which was also prevented by ARB (1.01 +/- 0.06). Renin qRTPCR in ANG II-infused rats showed higher mRNA levels in the kidney medulla compared with sham-operated rats (5.5 +/- 2.3 vs. 0.04 +/- 0.02 ratio to GAPDH mRNA levels; P < 0.001); however, renin transcript levels were normalized in the ARB-treated rats. These data demonstrate that the augmentation of distal nephron renin in ANG II-infused hypertensive rats is AT1 receptor mediated. The augmented distal tubular renin may contribute to increased intratubular ANG II levels and distal nephron sodium reabsorption in ANG II-dependent hypertension.

RETRACTED ARTICLE: Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats

Hypertension, endothelial dysfunction and insulin resistance are associated conditions that share oxidative stress and vascular inflammation as common features. Adiponectin is an abundant plasma adipokine that plays a physiological role in modulating lipid metabolism and exerts a potent anti-inflammatory activity. We hypothesised that adiponectin levels decrease in response to oxidative stress and that this may promote the development of hypertension, endothelial dysfunction and insulin resistance. Rats were infused with angiotensin II (AngII) or its vehicle, either alone or in combination with tempo1 (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a membrane-permeable metal-independent superoxide dismutase mimetic, or tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor for nitric oxide synthase activity. Heart rate, systolic blood pressure, body weight and serum levels of adiponectin were measured on day 7 of treatment, and then the animals were killed. Vessel tone and superoxide production were measured ex vivo in thoracic vascular rings. The expression of adiponectin mRNA in adipose tissue was assessed by Northern blotting, and in 3T3-L1 adipocytes exposed to H2O2 by real-time PCR. The expression of NAD(P)H oxidase subunit mRNAs in the rats was assessed by RT-PCR and real-time PCR. Hypertension and endothelial dysfunction were induced in rats by infusion of AngII and reversed by administration of tempol. Plasma concentrations of adiponectin and adipose tissue levels of adiponectin mRNA were decreased in AngII-infused rats, and this effect was prevented by cotreatment with tempol or BH4. The production of superoxide anions (O2-) was significantly increased in the aortae of AngII-treated rats, and this increase was prevented by the administration of tempol or BH4. Levels of mRNAs that encode NAD(P)H oxidase components, including p22phox, gp91phox, p47phox and Rac1, were similarly increased in adipose tissue, aortae and hearts of AngII-infused rats. Cotreatment of rats with tempol or BH4 reversed AngII-induced increases in NAD(P)H oxidase subunit mRNAs. Fully differentiated 3T3-L1 adipocytes, also exhibited diminished adiponectin mRNA levels when exposed to low concentrations of H2O2. Our results demonstrate that AngII-induced oxidative stress and endothelial dysfunction are accompanied by a decrease in adiponectin gene expression. Since antioxidants were observed to prevent the actions of AngII, and H2O2 on its own suppressed adiponectin expression, we conclude that adiponectin gene expression is negatively modulated by oxidative stress. Plasma adiponectin levels may provide a useful indicator of oxidative stress in vivo, and suppressed levels may contribute to the proinflammatory and metabolic derangements associated with type 2 diabetes, coronary artery disease and the metabolic syndrome.

Suppressors of Cytokine Signaling Regulate Angiotensin II-Activated Janus Kinase-Signal Transducers and Activators of Transcription Pathway in Renal Cells

Suppressors of cytokine signaling (SOCS) family is constituted by cytokine-inducible proteins that modulate receptor signal transduction via tyrosine kinases, mainly the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Differential SOCS expression was noted in renal cells that were incubated with inflammatory stimuli, but the role of SOCS in the pathogenesis of renal diseases is not yet well defined. Because angiotensin II (Ang II) plays a key role in renal disease, SOCS proteins were studied as a novel mechanism involved in the negative regulation of Ang II-mediated processes. Systemic Ang II infusion for 3 d increased the renal mRNA expression of SOCS-3 and SOCS-1. SOCS protein synthesis was found in glomerular mesangial area and tubules. In cultured mesangial cells and tubular epithelial cells, Ang II induced a rapid and transient SOCS-3 and SOCS-1 expression in parallel with JAK2 and STAT1 activation. In both cell types, overexpression of SOCS proteins prevented the STAT activation in response to Ang II. SOCS expression observed in Ang II-infused rats and in Ang II-stimulated cells was significantly inhibited by treatment with AT(1) but not AT(2) receptor antagonist and was attenuated in mesangial cells from AT(1a)-deficient mice, demonstrating the implication of AT(1) in those responses. In SOCS-3 knockdown studies, antisense oligonucleotides inhibited the expression of SOCS-3 and increased the Ang II-induced STAT activation and c-Fos/c-Jun expression, then resulting in a more severe renal damage. These results suggest that SOCS proteins may act as negative regulators of Ang II signaling in renal cells and implicate SOCS as important modulators of renal damage.

Effects of angiotensin-(1–7) blockade on renal function in rats with enhanced intrarenal Ang II activity

Increasing evidence suggests that angiotensin-(1-7) [Ang-(1-7)] acts as an endogenous antagonist of Ang II when the renin-angiotensin system (RAS) is activated. In the present study, we therefore compared the effects of acute intrarenal (i.r.) Ang-(1-7) receptor blockade on renal function under conditions of normal and increased intrarenal Ang II concentration. Salt-replete Hannover-Sprague Dawley rats (HanSD) served as control animals. As models with enhanced action of Ang II we first used transgenic rats harboring the Ren-2 renin gene (TGR), second, Ang II-infused rats, third, 2-kidney, 1-clip (2K1C) hypertensive rats on normal salt intake, and fourth, salt-depleted TGR and HanSD. I.r. Ang-(1-7) receptor blockade elicited significant decreases in glomerular filtration rate (GFR), renal plasma flow (RPF), and sodium excretion in 2K1C rats, and in salt-depleted TGR and HanSD. In contrast, i.r. Ang-(1-7) receptor blockade did not significantly change GFR, RPF, and sodium excretion in salt-replete TGR and HanSD, or in Ang II-infused rats. These findings suggest that under conditions of normal intrarenal RAS activity and increased intrarenal Ang II action by infusion of Ang II or by insertion of a renin gene in salt-replete conditions, Ang-(1-7) is not an important factor in the regulation of renal function. In contrast, under conditions of endogenous RAS activation due to clipping of the renal artery or to sodium restriction, Ang-(1-7) serves as opponent of the vasoconstrictor actions of Ang II.

Sympathetic Modulation by Carvedilol and Losartan Reduces Angiotensin II-Mediated Lipolysis in Subcutaneous and Visceral Fat

Advanced heart failure is characterized by increased activation of the renin-angiotensin system and the development of cachexia. Angiotensin II (Ang II) has been proposed as a lipid metabolism regulator. The effects of exogenous Ang II (osmotic minipump, 525 ng/kg/min for 12 d) on interstitial sc glycerol and norepinephrine levels, indexes of lipolysis, and sympathetic activation, respectively, were measured in Sprague Dawley rats by consecutive microdialysis performed in vivo in white adipose tissue. Higher sustained interstitial glycerol and norepinephrine levels were found after 7 and 12 d of Ang II infusion. Triglyceride to DNA content ratio and adipocyte diameter were reduced in sc and visceral (retroperitoneal and epididymal) fat tissues of Ang II-infused rats, whose body weight was lower and blood pressure higher. Losartan, an Ang II receptor 1 blocker, and carvedilol, an alpha1-nonselective-beta1,2,3-adrenergic blocker, but not doxazosin, an alpha1-selective-adrenergic blocker, lowered glycerol and norepinephrine levels, preventing lipolysis and weight loss. Our results indicate that Ang II stimulates lipolysis in sc and visceral adipocytes by sympathetic activation and beta-adrenergic-receptor stimulation. Nonselective-beta-adrenergic and Ang II-receptor1 blockade markedly attenuated the rise of norepinephrine, preventing catabolic effects. The metabolic benefits of carvedilol and losartan, in addition to recognized protective cardiovascular effects, may be relevant in cachectic patients with advanced heart failure.

Administration of ANG II induces iron deposition and upregulation of TGF-β1 mRNA in the rat liver

We previously found that ANG II infusion into rats causes iron deposition in the kidney and heart, which may have a role in the regulation of profibrotic gene expression and tissue fibrosis. In the present study, we have investigated whether ANG II can also induce iron accumulation in the liver. Prussian blue staining detected frequent iron deposition in the interstitium of the liver of rats treated with pressor dose ANG II for 7 days, whereas iron deposition was absent in the livers of control rats. Immunohistochemical and histological analyses showed that some iron-positive nonparenchymal cells were positive for ferritin and heme oxygenase-1 (HO-1) protein and TGF-beta1 mRNA and were judged to be monocytes/macrophages. It was shown that ANG II infusion caused about a fourfold increase in ferritin and HO-1 protein expression by Western blot analysis and about a twofold increase in TGF-beta1 mRNA expression by Northern blot analysis, which were both suppressed by treating ANG II-infused rats with losartan and deferoxamine. In addition, mild interstitial fibrosis was observed in the liver of rats that had been treated with pressor dose ANG II for 7 days or with nonpressor dose ANG II for 30 days, the latter of which also caused loss of hepatocytes and intrahepatic hemorrhage in the liver. Taken together, our data suggest that ANG II infusion induces aberrant iron homeostasis in the liver, which may have a role in the ANG II-induced upregulation of profibrotic gene expression in the liver.