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The 5/6th nephrectomy or ablation/infarction (A/l) preparation has been used as a classic model of chronic kidney disease (CKD). We observed increased kidney oxygen consumption (Qo2) and altered renal hemodynamics in the A/I kidney that were normalized after combined angiotensin II (ANG II) blockade. Studies suggest hypoxia inducible factor as a protective influence in A/I. We induced hypoxia-inducible factor (HIF) and HIF target proteins by two different methods, cobalt chloride (CO[Cl.sub.2]) and dimethyloxalyglycine (DMOG), for the first week after creation of A/I and compared the metabolic and renal hemodynamic outcomes to combined ANG II blockade. We also examined the HIF target proteins expressed by using Western blots and real-time PCR. Treatment with DMOG, CO[Cl.sub.2], and ANG II blockade normalized kidney oxygen consumption factored by Na reabsorption and increased both renal blood flow and glomerular filtration rate. At 1 wk, CO[Cl.sub.2] and DMOG increased kidney expression of HIF by Western blot. In the untreated A/I kidney, VEGF, heme oxygenase-1, and GLUT1 were all modestly increased. Both ANG II blockade and CO[Cl.sub.2] therapy increased VEGF and GLUT1 but the cobalt markedly so. ANG II blockade decreased heme oxygenase-1 expression while CO[Cl.sub.2] increased it. By real-time PCR, erythropoietin and GLUT1 were only increased by CO[Cl.sub.2] therapy. Cell proliferation was modestly increased by ANG II blockade but markedly after cobalt therapy. Metabolic and hemodynamic abnormalities were corrected equally by ANG II blockade and HIF therapies. However, the molecular patterns differed significantly between ANG II blockade and cobalt therapy. HIF induction may prove to be protective in this model of CKD. vascular endothelial growth factor; subtotal nephrectomy; heme oxygenase-1; kidney oxygen consumption doi: 10.1152/ajprenal.00153.2010

Accumulating evidence suggests that the intrarenal renin-angiotensin system may be involved in the progression of diabetic nephropathy. Chymase is a potent local angiotensin II-forming enzyme in several species, including humans and hamsters. However, the pathophysiological role of chymase is not fully understood. Here, we report a causal role of chymase in diabetic nephropathy and the therapeutic effectiveness of chymase inhibition. In the present study, renal chymase expression was markedly upregulated in streptozotocin-induced diabetic hamsters. Oral administration of a specific chymase inhibitor, TEI-F00806, completely ameliorated proteinuria, the overexpression of transforming growth factor-[beta] and fibronectin in glomeruli, and renal mesangial expansion, by normalizing the increase in intrarenal angiotensin II levels in diabetic hamsters independently of blood pressure levels. In contrast, ramipril did not show such sufficient effects. These effects occurred in parallel with improvements in superoxide production and expression of NAD(P)H oxidase components [NAD(P)H oxidase 4 and [p22.sup.phox]] in glomeruli. This study showed for the first time that chymase inhibition may protect against elevated intrarenal angiotensin II levels, oxidative stress, and renal dysfunction in diabetes. These findings suggest that chymase offers a new therapeutic target for diabetic nephropathy. angiotensin II; NAD(P)H oxidase doi: 10.1152/ajprenal.00337.2010

Endogenous and exogenous circulating ANG II acts at one of the central circumventricular organs (CVOs), the subfornical organ (SFO), to modulate chronic blood pressure regulation. However, at the forebrain, another important CVO is the organum vasculosum of the lamina terminalis (OVLT). In the present study, we tested the hypothesis that the OVLT mediates the hypertension or the hypotension produced by chronic infusion of ANG II or losartan (ATl antagonist), respectively. Six days after sham or OVLT electrolytic lesion, male Sprague-Dawley rats (280-320 g, n = 6 per group) were instrumented with intravenous catheters and radiotelemetric blood pressure transducers. Following another week of recovery, rats were given 3 days of saline control infusion (7 ml/day) and were then infused with ANG II (10 ng x [kg.sup.-1] x [min.sup.-1]) or losartan (10 mg x [kg.sup.-1] x [day.sup.-1]) for l0 days, followed by 3 recovery days. Twenty-four hour average measurements of mean arterial pressure (MAP) and heart rate (HR) were made during this protocol. Hydromineral balance (HB) responses were measured during the experimental protocol. By clay 9 of ANG II treatment, MAP had increased 16 [+ or -] 4 mmHg in sham rats but only 4 [+ or -] 1 mmHg in OVLT lesioned rats without changes in HR or HB. However, the hypotension produced by 10 days of losartan infusion was not modified in OVLT lesioned rats. These results suggest that the OVLT might play an important role during elevation of plasma ANG II, facilitating increases of blood pressure but is not involved with baseline effects of endogenous ANG II. hypertension; angiotensin II; losartan doi: 10.1152/ajpregu.00034.2010.

The role of angiotensin II (ANG II) in postnatal vasculogenesis and angiogenesis during skeletal muscle (SKM) regeneration is unknown. We examined the capacity of ANG II to stimulate capillary formation and growth during cardiotoxin-induced muscle regeneration in ACE inhibitor-treated ANG II type la receptor knockout ([AT1a.sup.-/-]) and C57Bl/6 control mice. Analysis of tibialis anterior (TA) cross-sections revealed 17% and 23% reductions in capillarization in [AT1a.sup.-/-] and captopril treated mice, respectively, when compared with controls, 21 days postinjury. Conversely, no differences in capillarization were detected at early time points (7 and 10 days). These results identify ANG II as a regulator of angiogenesis but not vasculogenesis in vivo. In vitro angiogenesis assays of human umbilical vein endothelial cells (HUVECs) further confirmed ANG II as proangiogeneic as 71% and 124% increases in tube length and branch point number were observed following ANG II treatment. Importantly, treatment of HUVECs with conditioned media from differentiated muscle cells resulted in an 84% and 203% increase in tube length and branch point number compared with controls, which was abolished following pretreatment of the cells with an angiotensin-converting enzyme inhibitor. The pro-angiogenic effect of ANG II can be attributed to an enhanced endothelial cell migration because both transwell and under agarose migration assays revealed a 37% and 101% increase in cell motility, respectively. Collectively, these data highlight ANG II as a proangiogenic regulator during SKM regeneration in vivo and more importantly demonstrates that ANG II released from SKM can signal endothelial cells and regulate angiogenesis through the induction of endothelial cell migration. captopril; migration; regeneration; vasculogenesis doi: 10.1152/ajpcell.00306.2010.

Angiotensin II (ANG II) contributes to hypertension, cardiac hypertrophy, fibrosis, and dysfunction; however, it is difficult to separate the cardiac effect of ANG II from its hemodynamic action in vivo. To overcome the limitations, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II) and treated them with deoxycorticosterone acetate (DOCA)-salt to suppress their systemic renin-angiotensin system. Using this unique model, we tested the hypothesis that cardiac ANG II, acting on the angiotensin type 1 receptor ([AT.sub.1]R), increases inflammation, oxidative stress, and apoptosis, accelerating cardiac hypertrophy and fibrosis. Male Tg-ANG II mice and their non-transgenic littermates (n-Tg) were uninephrectomized and divided into the following three groups: 1) vehicle-treated normotensive controls; 2) DOCA-salt; and 3) DOCA-salt + valsartan ([AT.sub.1]R blocker). Under basal conditions, systolic blood pressure (SBP) and cardiac phenotypes were similar between strains. In DOCA-salt hypertension, SBP increased similarly in both n-Tg and Tg-ANG II, and cardiac function did not differ between strains; however, Tg-ANG II had 1) greater ventricular hypertrophy as well as interstitial and perivascular fibrosis; 2) a higher number of deoxynucleotidyl-transferase-mediated dUTP nick end labeling-positive cells and infiltrating macrophages; 3) increased protein expression of NADPH oxidase 2 and transforming growth factor-[[beta].sub.1]; and 4) downregulation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (Akt) phosphorylation. Valsartan partially reversed these effects in Tg-ANG II but not in n-Tg. We conclude that, when hemodynamic loading conditions remain unchanged, cardiac ANG II does not alter heart size or cardiac functions. However, in animals with hypertension, cardiac ANG II, acting via [AT.sub.1]R, enhances inflammation, oxidative stress, and cell death (most likely via downregulation of PI 3-kinase and Akt), contributing to cardiac hypertrophy and fibrosis. inflammation; oxidative stress; cardiac hypertrophy; fibrosis doi: 10.1152/ajpheart.00538.2010.

It has been shown that angiotensin (ANG)-(1-7) activates nitric oxide synthase (NOS) in isolated ventricular myocytes from normotensive rats. Since many ANG-(1-7) actions are enhanced in situations of increased ANG II activity, as in hypertension, in this study we investigated the in vivo effect of ANG-(1-7) on NOS activity and expression of endothelial (eNOS), neuronal (nNOS), and inducible NOS (iNOS) in ventricles from spontaneously hypertensive rats (SHR). Rats were subjected to a 60-min ANG-(1-7) infusion (0.35 nmol/min); controls received saline. NOS activity was measured using the NADPH diaphorase histochemical method and by the conversion of L-[[sup.14]C]arginine to citrulline, and NOS phosphorylation and expression were determined using Western blotting. In SHR, ANG-(1-7) infusion diminished mean arterial pressure from 180 [+ or -] 9 to 146 [+ or -] 9 mmHg (P < 0.05), and this effect was prevented by nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. In addition, NOS activity and eNOS phosphorylation were increased by ANG-(1-7) infusion. Ventricular eNOS and nNOS expression were increased 67.4 [+ or -] 6.4 and 51 [+ or -] 10%, respectively, by ANG-(1-7), whereas iNOS was not changed. In another set of experiments, we evaluated the mechanism by which ANG-(1-7) modifies NOS activity. Isolated ventricle slices preincubated with ANG-(1-7) showed an increase in NOS activity and eNOS phosphorylation, which was blocked by an [AT.sub.2] and a bradykinin [B.sub.2] receptor antagonist, but not by the Mas receptor antagonist. Our results show that in rats in a hypertensive state, ANG-(1-7) infusion upregulates cardiac NOS expression and activity through an [AT.sub.2]- and bradykinin-dependent mechanism. In this way ANG-(1-7) may eficit its cardioprotective action and contribute to some of the counterregulatory [AT.sub.2] receptor effects that oppose the [AT.sub.1] receptor-mediated effects. receptors; hypertension; heart; bradykinin doi: 10.1152/ajpheart.00850.2009.

The study was undertaken to examine the potential cross talk between vasopressin and angiotensin II (ANG II) intracellular signaling pathways. We investigated in vivo and in vitro whether vasopressin-induced water reabsorption could be attenuated by ANG II AT1 receptor blockade (losartan). On a low-sodium diet (0.5 meq/day) dDAVP-treated animals with or without losartan exhibited comparable renal function [creatinine clearance 1.2 [+ or -] 0.1 in dDAVP + losartan (LSDL) vs. 1.1 [+ or -] 0.1 ml x 100 [g.sup.-1] x [day.sup.-1] in dDAVP alone (LSD), P > 0.05] and renal blood flow (6.3 [+ or -] 0.5 in LSDL vs. 6.8 [+ or -] 0.5 ml/min in LSD, P > 0.05). The urine output, however, was significantly increased in LSDL (2.5 [+ or -] 0.2 vs. 1.8 [+ or -] 0.2 ml x 100 [g.sup.-l] x [day.sup.-1], P < 0.05) in association with decreased urine osmolality (2,600 [+ or -] 83 vs. 3,256 [+ or -] 110 mosmol/kg[H.sub.2]O, P < 0.001) compared with rats in LSD. Immunoblotting revealed significantly decreased expression of medullary AQP2 (46 [+ or -] 6 vs. 176 [+ or -] 10% in LSD, P < 0.01), p-AQP2 (177 [+ or -] 13 vs. 214 [+ or -] 12% in LSD, P < 0.05), and AQP3 (134 [+ or -] 14 vs. 177 [+ or -] 11% in LSD, P < 0.05) in LSDL compared with LSD. The expressions of AQP1, the [[alpha].sub.1]- and [gamma]-subunits of Na-K-ATPase, and the Na-K-2Cl cotransporter were not different among groups. In vitro studies showed that ANG II or dDAVP treatment was associated with increased AQP2 expression and cAMP levels, which were potentiated by cotreatment with ANG II and dDAVP and were inhibited by AT1 blockade. In conclusion, ANG II AT1 receptor blockade in dDAVP-treated rats on a low-salt diet was associated with decreased urine concentration and decreased inner medullary AQP2, p-AQP2, and AQP3 expression, suggesting that AT1 receptor activation plays a significant role in regulating aquaporin expression and modulating urine concentration in vivo. Studies in collecting duct cells were confirmatory. aquaporin; urine concentration; cAMP doi: 10.1152/ajprenal.00168.2010.

To accelerate lung development and protect neonates from other early developmental problems, synthetic steroids are administered maternally in the third trimester, exposing fetuses that are candidates for premature delivery to them. However, steroid exposure at this point of gestation may lead to elevated blood pressure [mean arterial pressure (MAP)] during adolescence. We hypothesize that fetal exposure to steroids activates the renin-angiotensin system, inducing an elevation in blood pressure and attenuation of baroreflex sensitivity (BRS) that is angiotensin II dependent in early adulthood. To test this hypothesis, fetal sheep were exposed to betamethasone (Beta) or vehicle (control) administered to ewes at day 80 of gestation and delivered at full term. At 1.8 yr of age, male offspring were instrumented for conscious recording of MAP, heart rate, and measurement of BRS [as low-frequency-[alpha], high-frequency-co, sequence (seq) UP, seq DOWN, and seq TOTAL]. Beta-exposed sheep (n = 6) had higher MAP than control sheep (n = 5) (93 [+ or -] 2 vs. 84 [+ or -] 2 mmHg, P < 0.01). Acute blockade of angiotensin type 1 receptors with candesartan (0.3 mg/kg iv) normalized MAP in Beta-exposed sheep (85 [+ or -] 4 mmHg), with no effect in control sheep (82 [+ or -] 3 mmHg). Before angiotensin type 1 blockade, BRS maximum gain was significantly lower in Beta-exposed vs. control sheep (11 [+ or -] 3 vs. 26 [+ or -] 3 ms/mmHg, P < 0.0.01). However, 45 min after candesartan injection, BRS was increased in Beta-exposed (21 [+ or -] 5 ms/mmHg) and control (35 [+ or -] 4 ms/mmHg) sheep. Heart rate variability (HRV) and blood pressure variability (BPV) revealed lower HRV (SD of beat-to-beat interval and root mean square of successive beat-to-beat differences in R-R interval duration) and higher BPV (SD of MAP, systolic arterial pressure in low-frequency range) in Beta-exposed sheep. Candesartan partially restored HRV in Beta-exposed sheep and fully corrected BPV. Thus, in utero exposure to synthetic glucocorticoids causes long-lasting programming of the cardiovascular system via renin-angiotensin system-dependent mechanisms. spectral analysis; fetal programming; heart rate variability; blood pressure variability; renin-angiotensin system; hypertension doi: 10.1152/ajpheart.00100.2010.

Podocytes are an integral and important constituent of the glomerular filtration barrier (GFB) and are exposed to a higher concentrations of ANG II in diseased states; consequently, podocytes may accumulate oxidized proteins and damaged mitochondria. In the present study, we evaluated the effect of ANG II on the podocyte autophagic process, which is likely to be triggered in order to degrade unwanted proteins and damaged organelles. To quantitate the occurrence of autophagy, electron microscopic studies were carried out on control and ANG II-treated conditionally immortalized mouse podocytes (CIMPs). ANG II-treated cells showed a fivefold greater number of autophagosomes/field compared with control cells. This proautophagic effect of ANG II was inhibited by pretreatment with 3-methyladenine, an inhibitor of autophagy. ANG II also enhanced podocyte expression of autophagic genes such as LC3-2 and beclin-1. Since oxidative stress is often associated with the induction of autophagy, we examined the effect of ANG II on podocyte reactive oxygen species (ROS) generation. ANG II enhanced podocyte ROS generation in a time-dependent manner. To determine whether there is a causal relationship between ANG II-induced oxidative stress and induction of autophagy, we evaluated the effect of antioxidants on ANG II-induced autophagy. As expected, the proautophagic effect of ANG II was inhibited by antioxidants. We conclude that ANG II promotes podocyte autophagy through the generation of ROS. oxidative stress; angiotensin II doi: 10.1152/ajpcell.00424.2009.

Hyperglycemia is associated with hemodynamic changes in type 1 diabetes (DM), acting in part through renin-angiotensin system activation. Since aging is associated with vascular dysfunction in DM, we hypothesized that acute hemodynamic responses to clamped hyperglycemia and infused ANG II would be exaggerated in older adults compared with a group of adolescent/young adults with type 1 DM. Renal hemodynamic function, blood pressure, and arterial stiffness were assessed in adolescent/young adults (n = 34; mean age: 18 [+ or -] 3 yr) and older adults (n = 32; mean age: 45 [+ or -] 9 yr). Studies were performed during clamped euglycemia (4-6 retool/l) and hyperglycemia (9-11 mmol/ 1). Renal and systemic hemodynamic responses to ANG II were measured during clamped euglycemia in diabetic subjects. ANG II responses were also assessed in a cohort of non-DM subjects (n = 97; mean age: 26; age range: 18-40 yr). Older DM adults exhibited higher baseline blood pressure, arterial stiffness, and renal vascular resistance, and lower glomerular filtration rate (GFR) and effective renal plasma flow, compared with adolescent/young DM adults (P < 0.05). Clamped hyperglycemia was associated with exaggerated peripheral and renal hemodynamic responses uniquely in older DM adults; only GFR increased in adolescent/young DM adults. ANG II infusion also produced exaggerated vasoconstrictive responses in older DM adults vs. adolescent/young DM adults (P < 0.05). The independent effect of age on hemodynamic responses to hyperglycemia and ANG II was confirmed using multivariate regression analysis in DM subjects (P < 0.05), and results were still significant when participants were matched for DM duration. Age-related alterations in hemodynamic function and ANG II response were not observed in healthy non-DM control subjects. Acute hemodynamic responses to clamped hyperglycemia and ANG II were exaggerated in older subjects with type 1 DM, highlighting an important interaction between age and factors that contribute to the pathogenesis of acute vascular dysfunction in DM. renal hemodynamic function; systemic blood pressure; arterial stiffness doi: 10.1152/ajpregu.00027.2010.

Previous studies demonstrated that obesity increases inflammation in periaortic adipose tissue and promotes angiotensin II (ANG II)-induced abdominal aortic aneurysms (AAAs). We sought to determine whether weight loss of obese C57BL/6 mice would influence the progression of established AAAs. Male C57BL/6 mice were fed a high-fat diet (HF) for 4 mo and then infused with either saline or ANG II (1,000 ng x [kg.sup.-1] x [min.sup.-1]) for 3 mo. Mice with dilated suprarenal aortas at 28 days of ANG II infusion were designated to groups fed the HF (HF/HF) or a low-fat diet (LF; 10% kcal as fat; HF/LF) to induce weight loss for the last 2 mo of infusions. Suprarenal aortic lumen diameters of obese mice were increased by ANG II infusion at day 28 (day 0:1.03 [+ or -] 0.02; day 28:1.86 [+ or -] 0.14 mm; P < 0.05), but did not progress with continued infusion in HF/HF mice. Moreover, aortic lumen diameters were not different between groups (HF/HF: 1.89 [+ or -] 0.15; HF/LF: 1.79 [+ or -] 0.18 mm). However, maximal diameters of excised AAAs were decreased with weight loss (HF/HF: 2.00 [+ or -] 0.11; HF/LF: 1.55 [+ or -] 0.13 mm; P < 0.05) and had reduced adventitial areas (HF/HF: 1.18 [+ or -] 0.10; HF/LF: 0.54 [+ or -] 0.02 [mm.sup.2]; P < 0.05). Neovascularization of aortic adventitias was strikingly decreased in HF/LF mice (HF/HF: 43 [+ or -] 5; HF/LF: 12 [+ or -] 2 endothelial cells/ adventitial area; P < 0.05). ANG II-induced elevations in adipose mRNA abundance of CD105, an adipose-derived stem cell marker, were abolished with weight loss. These results demonstrate that weight loss limits adventitial expansion of ANG II-induced AAAs. Reduced neovascularization from weight loss may limit progression of AAAs. neovascularization; adipose stem cells doi: 10.1152/ajpheart.00961.2009.

The lungs are now recognized as an active metabolic organ that is a major determinant of the plasma concentrations of the vasoconstrictors endothelin (ET) and ANG II. Several studies have suggested a complex interaction between ET and ANG II in the systemic and coronary vascular beds that is different at rest and during exercise. To date, the interaction between these vasoconstrictor peptides has barely been investigated in relation to the pulmonary vascular bed. Consequently, we investigated the integrated control of pulmonary vasomotor tone by ET and ANG II in 24 chronically instrumented swine (15 female and 9 male) at rest and during graded treadmill exercise. In the systemic circulation, ANG II type 1 ([AT.sub.1]) receptor blockade with irbesartan and mixed [ET.sub.A]/[ET.sub.B] blockade with tezosentan each produced vasodilation. The systemic vasodilator effect of [ET.sub.A]/[ET.sub.B] blockade was enhanced after [AT.sub.1] blockade in female swine, whereas a trend toward an increase was observed in male swine. In the pulmonary circulation, [AT.sub.1] receptor blockade had no effect on pulmonary vascular tone in male swine, whereas it resulted in an unexpected increase in pulmonary vasomotor tone in female swine. [ET.sub.A]/[ET.sub.B] receptor blockade did not result in a decrease in pulmonary vasomotor tone at rest but produced a decrease in vasomotor tone during exercise in both genders. This pulmonary vasodilation by [ET.sub.A]/[ET.sub.B] receptor blockade was enhanced after prior [AT.sub.1] blockade in female swine but not in male swine. In conclusion, in both the systemic and pulmonary circulation of female swine, ANG II inhibits the vasoconstrictor influence of ET. This interaction is gender specific. The observation that plasma ET levels were not altered by [AT.sub.1] blockade in either gender suggests that the interaction between these vasoconstrictors occurs locally in the vasculature. microcirculation; exercise doi: 10.1152/ajpheart.00459.2009.

We have generated transgenic mice that express angiotensin II (ANG II) fused downstream of enhanced cyan fluorescent protein, expression of which is regulated by the mouse metallothionein promoter. The fusion protein, which lacks a secretory signal, is retained intracellularly. In the present study, RT-PCR, immunoblot analyses, whole-animal fluorescent imaging, and fluorescent microscopy of murine embryonic fibroblasts confirm expression of the fusion protein in vivo and in vitro. The transgene is expressed in all tissues tested (including brain, heart, kidney, liver, lung, and testes), and radioimmunoassay of plasma samples obtained from transgenic mice indicate no increase in circulating ANG II over wild-type levels, consistent with intracellular retention of the transgene product. Kidneys from transgenic and corresponding wild-type littermates were histologically evaluated, and abnormalities in transgenic mice consistent with thrombotic microangiopathy were observed; microthrombosis was frequently observed within the glomerular capillaries and small vessels. In addition, systolic and diastolic blood pressures, measured by telemetry (n = 8 for each group), were significantly higher in transgenic mice compared with wild-type littermates. Blood pressure of line A male transgenic mice was 125 [+ or -] 1.7 over 97 [+ or -] 1.6 compared with 109 [+ or -] 1.7 over 83 [+ or -] 1.4 mmHg in wild-type littermates (systolic over diastolic). In summary, overexpression of an intracellular fluorescent fusion protein of ANG II correlates with elevated blood pressure and kidney pathology. This transgenic model may be useful to further explore the intracellular renin-angiotensin system and its implication in abnormal kidney function and hypertension. intracrine; intracellular angiotensin; transgenic; renal microangiopathy doi: 10.1152/ajpheart.00027.2010.

We examined the mechanisms that maintain stable renal tissue [Po.sub.2] during moderate renal ischemia, when changes in renal oxygen delivery ([??]o.sub.2]) and consumption ([[??]o.sub.2]) are mismatched. When renal artery pressure (RAP) was reduced progressively from 80 to 40 mmHg, [[??]o.sub.2] (-38 [+ or -] 7%) was reduced more than [Do.sub.2] (-26 [+ or -] 4%). Electrical stimulation of the renal nerves (RNS) reduced [Do.sub.2] (-49 [+ or -] 4% at 2 Hz) more than [[??]o.sub.2] (-30 [+ or -] 7% at 2 Hz). Renal arterial infusion of angiotensin II reduced [Do.sub.2] (-38 [+ or -] 3%) but not [[??]o.sub.2] (+ 10 [+ or -] 10%). Despite mismatched changes in [[??]o.sub.2] and [[??]o.sub.2], renal tissue [Po.sub.2] remained remarkably stable at [greater than or equal to] 40 mmHg RAP, during RNS at [less than or equal to] 2 Hz, and during angiotensin II infusion. The ratio of sodium reabsorption to [[??]o.sub.2] was reduced by all three ischemic stimuli. None of the stimuli significantly altered the gradients in P[co.sub.2] or pH across the kidney. Fractional oxygen extraction increased and renal venous Po2 fell during 2-Hz RNS and angiotensin II infusion, but not when RAP was reduced to 40 mmHg. Thus reduced renal [[??]o.sub.2] can help. prevent tissue hypoxia during mild renal ischemia, but when renal [[??]o.sub.2] is reduced less than [[??]o.sub.2], other mechanisms prevent a fall in renal [Po.sub.2]. These mechanisms do not include increased efficiency of renal oxygen utilization for sodium reahsorption or reduced washout of carbon dioxide from the kidney, leading to increased oxygen extraction. However, increased oxygen extraction could be driven by altered countercurrent exchange of carbon dioxide and/or oxygen between renal arteries and veins. angiotensin II; carbon dioxide; hypoxia; hypotension; oxygen; sympathetic nervous system doi: 10.1152/ajprenal.00647.2009

Inflammatory factors are elevated in animal and human subjects with hypertension and renal injury. We hypothesized that inflammation contributes to hypertension-induced renal injury by impairing autoregulation and microvascular reactivity to [P2X.sub.1] receptor activation. Studies were conducted in vitro using the blood-perfused juxtamedullary nephron preparation. Rats receiving ANG II (60 ng/min) infusion were treated with the anti-inflammatory agent pentosan polysulfate (PPS) for 14 days. The magnitude and progression of hypertension were similar in ANG II and ANG II+PPS-treated rats (169 [+ or -] 5 vs. 172 [+ or -] 2 mmHg). Afferent arterioles from control rats exhibited normal autoregulatory behavior with diameter decreasing from 18.4 [+ or -] 1.6 to 11.4 [+ or -] 1.7 [micro]m when perfusion pressure was increased from 70 to 160 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in ANG II-treated rats, and diameter remained essentially unchanged over the range of perfusion pressures. However, ANG II-treated rats receiving PPS exhibited normal autoregulatory behavior compared with ANG II alone rats. Arteriolar reactivity to ATP and [beta],[gamma]-methylene ATP was significantly reduced in ANG II hypertensive rats compared with controls. Interestingly, PPS treatment preserved normal reactivity to P2 and [P2X.sub.1] receptor agonists despite the persistent hypertension. The maximal vasoconstriction was 79 [+ or -] 3 and 81 [+ or -] 2% of the control diameter for ATP and [beta], [gamma]-methylene ATP, respectively, similar to responses in control rats. PPS treatment significantly reduced [alpha]-smooth muscle actin staining in afferent arterioles and plasma transforming growth factor-[beta]1 concentration in ANG II-treated rats. In conclusion, PPS normalizes autoregulation without altering ANG II-induced hypertension, suggesting that inflammatory processes reduce [P2X.sub.1] receptor reactivity and thereby impair autoregulatory behavior in ANG II hypertensive rats. [P2X.sub.1] purinoceptor; afferent arteriole; pentosan polysulfate doi: 10.1152/ajprenal.00743.2009.

Contag SA, Bi J, Chappell MC, Rose JC. Developmental effect of antenatal exposure to betamethasone on renal angiotensin H activity in the young adult sheep. Am J Physiol Renal Physiol 298: F847-F856, 2010. First published January 13, 2010; doi:10.1152/ajprenal.00497.2009.--Antenatal corticosteroids may have long-term effects on renal development which have not been clearly defined. Our objective was to compare the responses to intrarenal infusions of ANG II in two groups of year-old, male sheep: one group exposed to a clinically relevant dose of betamethasone before birth and one not exposed. We wished to test the hypothesis that antenatal steroid exposure would enhance renal responses to ANG II in adult life. Six pairs of male sheep underwent unilateral nephrectomy and renal artery catheter placement. The sheep were infused for 24 h with ANG II or with ANG II accompanied by blockade of the angiotensin type 1 (A[T.sub.1]) or type 2 (AT2) receptor. Baseline mean arterial blood pressure among betamethasone-exposed sheep was higher than in control animals (85.8 [+ or -] 2.2 and 78.3 [+ or -] 1.0 mmHg, respectively, P = 0.003). Intrarenal infusion of ANG II did not increase systemic blood pressure (P [greater than or equal to] 0.05) but significantly decreased effective renal plasma flow and increased renal artery resistance (P < 0.05). The decrease in flow and increase in resistance were significantly greater in betamethasone- compared with vehicleexposed sheep (betamethasone P < 0.05, vehicle P [greater than or equal to] 0.05). This effect appeared to be mediated by a heightened sensitivity to the A[T.sub.1] receptor among betamethasone-exposed sheep. Sodium excretion initially decreased in both groups during ANG II infusion; however, a rebound was observed after 24 h. A[T.sub.1] blockade was followed by a significant rebound after 24 h in both groups. A[T.sub.2] blockade blunted the 24-h rebound effect among the vehicle-exposed sheep compared with the betamethasone-exposed sheep. In conclusion, antenatal corticosteroid exposure appears to modify renal responsiveness to ANG II by increasing A[T.sub.1]- and decreasing A[T.sub.2] receptor-mediated actions particularly as related to renal blood flow and sodium excretion. angiotensin receptor; renal plasma flow; glomerular filtration rate; corticosteroids doi: 10.1152/ajprenal.00497.2009.

Crowley SD, Song Y, Lin EE, Griffiths R, Kim H, Ruiz P. Lymphocyte responses exacerbate angiotensin H-dependent hypertension. Am J Physiol Regul Integr Comp Physiol 298: R1089-R1097, 2010. First published February 10, 2010; doi:10.1152/ajpregu.00373.2009.--Activation of the immune system by ANG II contributes to the pathogenesis of hypertension, and pharmacological suppression of lymphocyte responses can ameliorate hypertensive end-organ damage. Therefore, to examine the mechanisms through which lymphocytes mediate blood pressure elevation, we studied ANG II-dependent hypertension in scid mice lacking lymphocyte responses and wild-type controls. Scid mice had a blunted hypertensive response to chronic ANG II infusion and accordingly developed less cardiac hypertrophy. Moreover, lymphocyte deficiency led to significant reductions in heart and kidney injury following 4 wk of angiotensin. The muted hypertensive response in the scid mice was associated with increased sodium excretion, urine volumes, and weight loss beginning on day 5 of angiotensin infusion. To explore the mechanisms underlying alterations in blood pressure and renal sodium handling, we measured gene expression for vasoactive mediators in the kidney after 4 wk of ANG II administration. Scid mice and controls had similar renal expression for interferon-[gamma], interleukin-1[beta], and interleukin-6. By contrast, lymphocyte deficiency (i.e., scid mice) during ANG II infusion led to upregulation of tumor necrosis factor-[alpha], endothelial nitric oxide synthase (eNOS), and cyclooxygenase-2 (COX-2) in the kidney. In turn, this enhanced eNOS and COX-2 expression in the scid kidneys was associated with exaggerated renal generation of nitric oxide, prostaglandin [E.sub.2], and prostacyclin, all of which promote natriuresis. Thus, the absence of lymphocyte activity protects from hypertension by allowing blood pressure-induced sodium excretion, possibly via stimulation of eNOSand COX-2-dependent pathways. inflammation; kidney diseases; T lymphocytes doi: 10.1152/ajpregu.00373.2009.

Previous studies have shown that in proximal and distal tubule nephron segments, peritubular ANG II stimulates sodium chloride transport. However, ANG II inhibits chloride transport in the medullary thick ascending limb (mTAL). Because ANG II and catecholamines are both stimulated by a decrease in extracellular fluid volume, the purpose of this study was to examine whether there was an interaction between ANG II and catecholamines to mitigate the inhibition in chloride transport by ANG II. In isolated perfused rat mTAL, [10.sup.-8] M bath ANG II inhibited wansport (from a basal transport rate of 165.6 [+ or -] 58.8 to 58.8 [+ or -] 29.4 pmol*[mm.sup.-1]*[min.sup.-1]; p < 0.0l). Bath norepinephrine stimulated chloride transport (from a basal transport rate of 298.1 [+ or -] 31.7 to 425.2 [+ or -] 45.8 pmol*[mm.sup.-1]*[min.sup.-1]; P < 0.05) and completely prevented the inhibition in chloride transport by ANG II. The stimulation of chloride transport by norepinephrine was mediated entirely by its [beta]-adrenergic effect; however, both the [beta]- and [alpha]-adrenergic agonists isoproterenol and phenylephrine prevent the ANG II-mediated inhibition in chloride transport. In the presence of [10.sup.-5] M propranolol, the effect of norepinephrine to prevent the inhibition of chloride transport by ANG II was still present. These data are consistent with an interaction of both [alpha]- and [beta]-catecholamines and ANG II on net chloride transport in the mTAL. NKCC2; norepinephrine; isoproterenol doi: 10.1152/ajpregu.00758.2009.

Recent studies have shown that asymmetric dimethylarginine (ADMA), a nitric oxide synthase inhibitor, is increased in hypertension and chronic kidney disease. However, little is known about the effects of hypertension per se on ADMA metabolism. The purpose of this study was to test the hypothesis that ANG II-induced hypertension, in the absence of renal injury, is associated with increased oxidative stress and plasma and renal cortex ADMA levels in rats. Male Sprague-Dawley rats were treated with ANG II at 200 ng x [kg.sup.-1] x [min.sup.-1] sc (by minipump) for 1 or 3 wk or at 400 ng x [kg.sup.-1] x [min.sup.-1] for 6 wk. Mean arterial pressure was increased after 3 and 6 wk of ANG II; however, renal injury (proteinuria, glomerular sclerosis, and interstitial fibrosis) was only evident after 6 wk of treatment. Plasma thiobarbituric acid reactive substances concentration and renal cortex [p22.sup.phox] protein abundance were increased early (1 and 3 wk), but urinary excretion of isoprostane and [H.sub.2][O.sub.2] was only increased after 6 wk of ANG II. An increased in plasma ADMA after 6 wk of ANG II was associated with increased lung protein arginine methyltransferase-1 abundance and decreased renal cortex dimethylarginine dimethylaminohydrolase activity. No changes in renal cortex ADMA were observed. ANG II hypertension in the absence of renal injury is not associated with increased ADMA; however, when the severity and duration of the treatment were increased, plasma ADMA increased. These data suggest that elevated blood pressure alone, for up to 3 wk, in the absence of renal injury does not play an important role in the regulation of ADMA. However, the presence of renal injury and sustained hypertension for 6 wk increases ADMA levels and contributes to nitric oxide deficiency and cardiovascular disease. kidney; protein arginine methyltransferase-1; dimethylarginine dimethylaminohydrolase; oxidative stress doi:10.1152/ajpregu.90875.2008

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/ rain) in the presence or absence of candesartan (1 [mu]M), PD-123319 (1 [mu]M), and rosiglitazone (0.1 [mu]M). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of ATI receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant -50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-[AT.sub.1] mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and [AT.sub.1] receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-[gamma] agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretchinduced activation of the angiotensin II-[AT.sub.1] receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented [AT.sub.1] upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-[gamma] agonists in the prevention of this loss. diabetic nephropathy; proteinuria; [AT.sub.1] receptor doi: 10.1152/ajprenal.90423.2008.

Neuropeptide Y (NPY) is a cotransmitter with norepinephrine (NE) and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin-angiotensin system (RAS), as well as a role for NPY in the development of hypertension in experimental animal models and in humans. Angiotensin II (ANG II) is known to facilitate sympathetic neurotransmission, an effect greater in spontaneously hypertensive rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. A newly discovered product of the RAS is angiotensin-(1-7) [ANG-(I-7)]. There is evidence suggesting that ANG-(1-7) opposes the actions of ANG II, resulting in hypotensive effects. The objective of this study was to investigate the role of ANG-(1-7) on the nerve-stimulated overflow of NE and NPY from the mesenteric arterial bed of SHR and the mechanisms involved in mediating any effects produced. ANG-(1-7) (0.001, 0.01, 0.1 [micro]M) decreased nerve-stimulated NE and NPY overflow, as well as perfusion pressure in preparations obtained from SHR. This effect was greater in preparations of SHR than WKY controls. In addition, ANG-(1-7) decreased NE overflow to a greater extent than NPY overflow. Administration of the Mas receptor antagonist, D-Ala7 ANG-(1-7), attenuated the decrease in both NE and NPY overflow due to ANG-(1-7) administration. However, the angiotensin type 2 receptor antagonist, PD-123391, attenuated the effect of ANG-(1-7) on NE overflow without affecting the decrease in NPY overflow. Moreover, in the presence of [N.sup.G]-nitro-L-arginine methyl ester, ANG(1-7) decreased NPY overflow, but not NE overflow. ANG-(1-7) decreases the nerve-stimulated overflow of NE and NPY in preparations of SHR, whereas ANG II enhances it. Therefore, ANG-(I-7) may counteract the effects of ANG II by acting on ANG type 2 and Mas receptors. sympathetic neurotransmission; nitric oxide; bradykinin; mesenteric artery doi: 10.1152/ajpheart.00400.2009

Mizushima T, Sasaki M, Ando T, Wada T, Tanaka M, Okamoto Y, Ebi M, Hirata Y, Murakami K, Mizoshita T, Shimura T, Kubota E, Ogasawara N, Tanida S, Kataoka H, Kamiya T, Alexander JS, Joh T. Blockage of angiotensin II type 1 receptor regulates TNF-[alpha]-induced MAdCAM-1 expression via inhibition of NF-[kappa]B translocation to the nucleus and ameliorates colitis. Am J Physiol Gastrointest Liver Physiol 298: G255-G266, 2010. First published November 25, 2009; doi: 10.1152/ajpgi.00264.2009.-- Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is an important target in the treatment of inflammatory bowel disease (1BD). Recently, treatment of IBD with an antibody to [alpha]4137-integrin, a ligand for MAdCAM-1, has been an intense focus of research. Our aim was to clarify the mechanism by which MAdCAM-1 is regulated via angiotensin II type 1 receptor (AT1R), and to verify if AT1R might be a novel target for IBD treatment. The role of AT1R in the expression of MAdCAM-1 in SVEC (a murine high endothelial venule cell) and MJC-1 (a mouse colonic endothelial cell) was examined following cytokine stimulation. We further evaluated the effect of AT1R on the pathogenesis of immunemediated colitis using AT1R-deficient (AT1R-/-) mice and a selective AT1R blocker. AT1R blocker significantly suppressed MAdCAM-1 expression induced by TNF-[alpha], but did not inhibit phosphorylation of p38 MAPK or of I[kappa]B that modulate MAdCAM-1 expression. However, NF-[kappa]B translocation into the nucleus was inhibited by these treatments. In a murine colitis model induced by dextran sulfate sodium, the degree of colitis, judged by body weight loss, histological damage, and the disease activity index, was much milder in AT1R-/- than in wild-type mice. The expression of MAdCAM-I was also significantly lower in AT1R-/- than in wild-type mice. These results suggest that AT1R regulates the expression of MAdCAM-1 under colonic inflammatory conditions through regulation of the translocation of NF-[kappa]B into the nucleus. Furthermore, inhibition of ATIR ameliorates colitis in a mouse colitis model. Therefore, AT1R might be one of new therapeutic target of IBD via regulation of MAdCAM-1. mucosal addressin cell adhesion molecule-l; inflammatory bowel disease; nuclear factor-[kappa]B doi: 10.1152/ajpgi.00264.2009

Am J Physiol Renal Physiol 298: F177-F186, 2010. First published October 28, 2009; doi:10.1152/ajprenal.00464.2009.--Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the [Na.sup.+]/[H.sup.+] exchanger isoform 3 (NHE3), [Na.sup.+]/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 [micro]g/min for 20 min) that increased PT flow rate ~20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng x [kg.sup.-1] x [min.sup.-1] for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG n included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar [H.sup.+]-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption. hypertension; captopril

Am J Physiol Renal Physiol 298: F125-F132, 2010. First published October 28, 2009; doi: 10.1152/ajprenal.00248.2009.--Glomerular endothelial cells (GEC) are strategically situated within the capillary loop and adjacent to the glomerular mesangium. GEC serve as targets of metabolic, biochemical, and hemodynamic signals that regulate the glomerular microcirculation. Unequivocally, hyperglycemia, hypertension, and the local renin-angiotensin system partake in the initiation and progression of diabetic nephropathy (DN). Whether free fatty acids (FFA) and reactive oxygen species (ROS) that have been associated with the endothelial dysfunction of diabetic macrovascular disease also contribute to DN is not known. Since endothelial cells from different organs and from different species may display different phenotypes, we employed human GEC to investigate the effect of high glucose (22.5 mmol/l), FFA (800 [micro]mol/l), and angiotensin II (ANG II; [10.sup.-7] mol/l) on the genesis of ROS and their effects on endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2), and the synthesis of prostaglandins (PGs). We demonstrated that high glucose but not high FFA increased the expression of a dysfunctional eNOS as well as increased ROS from NADPH oxidase (100%) and likely from uncoupled eNOS. ANG II also induced ROS from NADPH oxidase. High glucose and ANG II upregulated (100%) COX-2 via ROS and significantly increased the synthesis of prostacyclin ([PGI.sub.2]) by 300%. In contrast, FFA did not upregulate COX-2 but increased [PGI.sub.2] (500%). These novel studies are the first in human GEC that characterize the differential role of FFA, hyperglycemia, and ANG II on the genesis of ROS, COX-2, and PGs and their interplay in the early stages of hyperglcyemia. diabetes; prostaglandins; reactive oxygen species; nitric oxide

Stec DE, Vera T, Storm MV, McLemore GR Jr, Ryan MJ. Blood pressure and renal blow flow responses in heme oxygenase-2 knockout mice. Am J Physiol Regul Integr Comp Physiol 297: R1822-R1828, 2009. First published October 21, 2009; doi: 10.1152/ajpregu.00319.2009.--Heme oxygenase (HO) is the enzyme responsible for the breakdown of heme-generating carbon monoxide (CO) and biliverdin in this process. HO-2 is the constitutively expressed isoform in most tissues, such as the kidney and vasculature. CO generated by HO is believed to be an important vasodilator in the renal circulation along with another gas, nitric oxide (NO). To determine the importance of HO-2 in the regulation of blood pressure and renal blood flow (RBF), we treated HO-2 knockout (KO) mice chronically with either ANG II or [N.sup.G]-nitroarginine methyl ester (L-NAME). Basal blood pressures were not different between wild-type (WT), heterozygous (HET), or KO mice and averaged 113 [+ or -] 3 vs. 115 [+ or -] 2 vs. 116 [+ or -] 2 mmHg. Similar increases in blood pressure to chronic ANG II as well as L-NAME treatment were observed in all groups with blood pressures increasing an average of 30 mmHg in response to ANG II and 15 mmHg in response to L-NAME. Basal RBFs were not different between the groups averaging 6.0 [+ or -] 0.5 (n = 6) vs. 4.8 [+ or -] 0.6(n= 10) vs. 5.8 [+ or -] 0.7 (n = 6) ml x [min.sup.-1] x [g.sup.-1] kidney weight in WT, HET, and KO mice. HO-2 KO and HET mice exhibited an attenuated decrease in RBF in response to acute administration of ANG II, while no differences were observed with L-NAME. Our data indicate that blood pressure and RBF responses to increased ANG II or inhibition of nitric oxide are not significantly enhanced in HO-2 KO mice. angiotensin II; nitric oxide: L-NAME; heine oxygenase-1; carbon monoxide; renal vascular resistance; gene targeting doi: 10.1152/ajpregu.00319.2009

Lutken SC, Kim SW, Jonassen T, Marples D, Knepper MA, Kwon TH, Frokiaer J, Nielsen S. Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II [AT.sub.1] receptor blockade. Am J Physiol Renal Physiol 297: F1678-F1688, 2009. First published September 23, 2009; doi: 10.1152/ajprenal.00010.2009.--Heart failure (HF) was induced by ligation of the left anterior descending artery (LAD). Left ventricular end-diastolic pressure (LVEDP) >25 mmHg (at day 23 after LAD ligation) was the inclusion criterion. The rats were divided into three groups: sham-operated (Sham, n = 23, LVEDP: 5.6 [+ or -] 0.6 mmHg), HF (n = 14, LVEDP: 29.4 [+ or -] 1.4 mmHg), and candesartan (1 mg x [kg.sup.-1] [day.sup.-1] sc)-treated HI= (HF + Can, n = 9, LVEDP: 29.2 [+ or -] 1.2 mmHg). After 7 days (i.e., 29 days after LAD ligation) semiquantitative immunoblotting revealed increased abundance of inner medulla aquaporin-2 (AQP2) and AQP2 phosphorylated at [Ser.sup.256] (p-AQP2) in HF. There was also markedly enhanced apical targeting of AQP2 and p-AQP2 in inner medullary collecting duct (IMCD) in HF compared with Sham rats, shown by immunocyto-chemistry. Candesartan treatment significantly reversed the increases in both AQP2 and p-AQP2 expression and targeting. In contrast, there were only modest changes in other collecting duct segments. Semiquantitative immunoblots revealed increased expression of type 3 [Na.sup.+]/[H.sup.+] exchanger (NHE3) and [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2) in kidneys from HF compared with Sham rats: both effects were reversed or prevented by candesartan treatment. The protein abundance of [alpha]-epithelial sodium channel ([alpha]-ENaC) was increased while [beta]-ENaC and [gamma]-ENaC expression was decreased in the cortex and outer stripe of the outer medulla in HF compared with Sham rats, which was partially reversed by candesartan treatment. These findings strongly support an important role of angiotensin II in the pathophysiology of renal water and sodium retention associated with HF. collecting duct: aquaporin; type 3 [Na.sup.+]/[H.sup.+] exchanger; [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter; epithelial sodium channel doi: 10.1152/ajprenal.00010.2009

Only few studies have been devoted to the actions of the renin-angiotensin system (RAS) in the human gastrointestinal tract. The present study was undertaken to elucidate the expression and action of RAS in the human esophageal mucosa. Mucosal specimens with normal histological appearance were obtained from healthy subjects undergoing endoscopy and from patients undergoing esophagectomy due to neoplasm. Gene and protein expressions of angiotensin II (Ang II) receptor type 1 ([AT.sub.1]) and type 2 ([AT.sub.2]) and angiotensin-converting enzyme (ACE) were analyzed. In vivo functionality in healthy volunteers was reflected by assessing transmucosal potential difference (PD). Ussing chamber technique was used to analyze the different effects of Ang II on its [AT.sub.1] and [AT.sub.2] receptors. Immunoreactivity to [AT.sub.1] and [AT.sub.2] was localized to stratum superficiale and spinosum in the epithelium. ACE, [AT.sub.1], and [AT.sub.2] were found in blood vessel walls. Transmucosal PD in vivo increased following administration of the [AT.sub.1] receptor antagonist candesartan. In Ussing preparations mean basal transmural PD was -6.4 mV, epithelial current ([I.sub.ep]) 34 [micro]A/[cm.sup.2], and epithelial resistance ([R.sub.ep]) 321 [ohms] x [cm.sup.2]. Serosal exposure to Ang II increased PD as a result of increased [I.sub.ep], whereas [R.sub.ep] was constant. Ang II given together with the selective [AT.sub.1]-receptor antagonist losartan, or [AT.sub.2] agonist C21 given alone, resulted in a similar effect. Ang II given in presence of the [AT.sub.2]-receptor antagonist PD123319 did not influence PD, but [I.sub.ep] decreased and [R.sub.ep] increased. In conclusion, Ang II receptors and ACE are expressed in the human esophageal epithelium. The results suggest that [AT.sub.2]-receptor stimulation increases epithelial ion transport, whereas the [AT.sub.1] receptor inhibits ion transport and increases [R.sub.ep]. potential difference; epithelial current; epithelial resistance doi: 10.1152/ajpgi.00255.2009.

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via [AT.sub.1] ([AT.sub.1a]) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates [AT.sub.1]-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of [AT.sub.1] receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before [AT.sub.1]-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. [AT.sub.1] siRNAs inhibited [AT.sub.1] expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by ~90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited [AT.sub.1]-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in ~52% decreases in [AT.sub.1]-mediated FITC-ANG II uptake and ~66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered [AT.sub.1] expression or clathrin-dependent transferrin uptake. Both losartan and [AT.sub.1a] receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in [AT.sub.1] ([AT.sub.1a])-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells. kidney; clathrin-coated pits; cytoskeleton; G protein-coupled receptor; MAP kinases; small interfering RNA doi: 10.1152/ajprenal.90734.2008.

To investigate the role of brain angiotensin II (ANG II) in the pathogenesis of injury following ischemic stroke, mice overexpressing renin and angiotensinogen (R + A +) and their wild-type control animals (R - A -) were used for experimental ischemia studies. Focal brain ischemia was induced by middle cerebral artery occlusion (MCAO). The severity of ischemic injury was determined by measuring neurological deficits and histological damage at 24 and 48 h after MCAO, respectively. To exclude the influence of blood pressure and local collateral blood flow, brain slices were used for oxygen and glucose deprivation (OGD) studies. The severity of OGD-induced damage was determined by measuring indicators of tissue swelling and cell death, the intensity of the intrinsic optical signal (IOS), and the number of propidium iodide (PI) staining cells, respectively. Results showed 1) R + A + mice showed higher neurological deficit score (3.8 [+ or -] 0.5 and 2.5 [+ or -] 0.3 for R + A + and R - A -, respectively, P < 0.01) and larger infarct volume (22.2 [+ or -] 1.6% and 14.1 [+ or -] 1.2% for R + A + and R - A -, respectively, P < 0.01); 2) The R + A + brain slices showed more severe tissue swelling and cell death in the cortex (IOS: 140 [+ or -] 6% and 114 [+ or -] 10%; PI: 139 [+ or -] 20 cells/field and 39 [+ or -] 9 cells/field for R + A + and R - A -, respectively, P < 0.01); 3) treatment with losartan (20 [micro]mol/1) abolished OGD-induced exaggeration of cell injury seen in R + A + mice. The data indicate that activation of ANG II/[AT.sub.1] signaling is harmful to brain exposed to ischemia. [AT.sub.1] receptor; mouse; losartan; middle cerebral artery doi: 10.1152/ajpregu.91040.2008.

Ischemic focal ventricular tachycardia (VT) occurs in animals and humans. Angiotensin-converting enzyme inhibitors and receptor blockers reduce sudden death in patients with ischemic heart disease. In our dog model of coronary artery occlusion (CAO), we tested the hypothesis that angiotensin II (AGII) will selectively promote focal VT and that the specific [AT.sub.2] blocker PD-123319 (PD), or ATI blocker losartan, will affect this VT. Anesthetized dogs (n = 90) underwent CAO, followed by three-dimensional activation mapping of inducible VT. Dogs without VT in 1-3 h after CAO received AGII, and those with VT received either PD or losartan. Focal endocardium excised from ischemic sites was studied in vitro with standard microelectrode. Of 33 dogs with no inducible VT, AGII infusion resulted in sustained VT of only focal Purkinje origin in 13 (39%) compared with 0 of 20 dogs with saline. Of 26 dogs with inducible VT at baseline, given PD, reinduction was blocked in 8 of 10 (P < 0.05) focal VT, but only 1 of 15 with reentry. In contrast, of 11 dogs given losartan, reinduction of either mechanism was not blocked. In vitro triggered activity in Purkinje was blocked by PD in 13 of 19 (P < 0.05), but not by losartan in 8. Also, triggered activity was promoted by AGII, losartan, or the combination in 9 of 12 tissues. AGII promotes only focal, mainly Purkinje ischemic VT. PD, but not losartan, preferentially blocked focal VT, which is likely due to triggered activity due to delayed afterdepolarizations in Purkinje. triggered activity; canine model; lorsartan doi: 10.1152/ajpheart.00080.2009.

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 ([AT.sub.1]) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh ([10.sup.-9]-[10.sup.-5] M), iloprost ([10.sup.-16]-[10.sup.-11] g/ml), and reduced P[Osub.2] was lost and the ratio of phospho-ERK/ERKl/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ng x [kg.sup.-1] x [min.sup.-1]) or epidermal growth factor (EGF; 2 [micro]g/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG- 1478 (20 [micro]g/h), the ERK 1/2 inhibitor PD-98059 (10 [micro]g/h), or the protein synthesis inhibitor cycloheximide (5 [micro]g/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced P[O.sub.2], and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced P[O.sub.2] and iloprost were unaffected by the p38 MAP kinase inhibitor SB203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2. hypertension; salt: cell signaling; oxidative stress; renin-angiotensin system; epidermal growth factor; extracellular signal-regulated kinase doi: 10.1152/ajpheart.01325.2008

The effects of chronic mild hypoxemia on the binding of angiotensin receptors in selected brain stem nuclei and reflex responses were studied in fetal sheep. Fetal and maternal catheters were placed at 120 days' gestation, and animals received intratracheal maternal administration of nitrogen (n = 16) or compressed air in controls (n = 19). Nitrogen infusion was adjusted to reduce fetal brachial artery P[O.sub.2] by 25% during 5 days. Spontaneous baroreflex sensitivity and spectral analysis of the pulse interval were analyzed during the 5 days hypoxemia period using 90 min of daily recording. Brains of control and hypoxemic animals were collected, and brain stem angiotensin receptor binding was studied by in vitro autoradiography at 130 days of gestation. After 5 days of hypoxemia, some animals in each group were submitted to one complete umbilical cord occlusion during 5 min. [[sup.125]I]sarthran binding showed that chronic mild hypoxemia significantly increases angiotensin type 1 receptor, angiotensin type 2 receptor, and ANG-(1-7) angiotensin receptor binding sites in the nucleus tractus solitarius and dorsal motor nucleus of the vagus (P < 0.05). Hypoxemia induced lower baroreflex sensitivity and a higher low frequency-to-high frequency ratio in the fetus, consistent with a shift from vagal to sympathetic autonomic cardiac regulation. Cord occlusion to elicit a chemoreflex response induced a greater bradycardic response in hypoxemic fetuses (slope of the initial fall in heart rate; 11.3 [+ or -] 1.9 vs. 6.4 [+ or -] 1.2 beats x [min.sup.-1] x [s.sup.-1], P < 0.05). In summary, chronic mild hypoxemia increased binding of angiotensin receptors in brain stem nuclei, decreased spontaneous barorefex gain, and increased chemoreflex responses to asphyxia in the fetus. These results suggest hypoxemia-induced alterations in brain stem mechanisms for cardiovascular control. fetus; hypoxemia; baroreflex

Expression of nuclear angiotensin II type 1 ([AT.sub.1]) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist [sup.125]I-[[Sar.sup.1],[Thr.sup.8]]-ANG II ([sup.125]I-sarthran) with the [AT.sub.1] antagonist losartan (LOS) or the [AT.sub.2] antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC ([greater than or equal to] 70%) and PM ([greater than or equal to]80%) sites while LOS competition predominated in medullary NUC ([greater than or equal to]75%) and PM ([greater than or equal to]70%). Immunodetection with an [AT.sub.2] antibody revealed a single ~42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized [AT.sub.2] in the tubulointerstitium, [AT.sub.1] in the medulla and vasa recta, and both [AT.sub.1] and [AT.sub.2] in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-L-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear [AT.sub.2] receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure. nucleus; kidney; intracellular renin angiotensin system

Recent evidence suggests that mineralocorticoid receptor (MR) antagonism has beneficial effects on tissue oxidative stress and insulin metabolic signaling as well as reducing proteinuria. However, the mechanisms by which MR antagonism corrects both renin-angiotensin-aldosterone system (RAAS) impairments in renal insulin metabolic signaling and filtration barrier/podocyte injury remain unknown. To explore this potential beneficial interactive effect of MR antagonism we used young transgenic (mRen2)27 (Ren2) rats with increased tissue RAAS activity and elevated serum aldosterone levels. Ren2 and age-matched Sprague-Dawley (SD) control rats (age 6-7 wk) were implanted with a low dose of the MR antagonist spironolactone (0.24 mg/day) or vehicle, both delivered over 21 days. Albuminuria, podocyte-specific proteins (synaptopodin, nephrin, and podocin), and ultrastructural analysis of the glomerular filtration barrier were measured in relation to RAAS activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reactive oxygen species (ROS), and the redox-sensitive Rho kinase (ROK). Insulin metabolic signaling was determined via measurement of insulin receptor substrate-1 (IRS-1) phosphorylation, IRS-1 ubiquitin/proteasomal degradation, and phosphorylation of Akt. Ren2 rats exhibited albuminuria, loss of podocytespecific proteins, and podocyte foot process effacement contemporaneous with reduced renal IRS-1 and protein kinase B/Akt phosphorylation compared with SD control rats (each P < 0.05). Ren2 kidneys also manifested increased NADPH oxidase/ROS/ROK in conjunction with enhanced renal tissue levels of angiotensin II (ANG II), ANG(1-12), and angiotensin type 1 receptor. Low-dose spironolactone treatment reduced albuminuria and tissue RAAS activity and improved podocyte structural and protein integrity with improvements in IRS-1/Akt phosphorylation. Thus, in this model of RAAS activation, MR antagonism attenuates glomerular/podocyte remodeling and albuminuria, in part through reductions in redox-mediated impairment of insulin metabolic signaling. renal mineralocorticoid receptor; reduced nicotinamide adenine dinucleotide phosphate oxidase; oxidative stress; podocyte

In angiotensin II (ANG II)-induced hypertension, intrarenal ANG II levels are increased by [AT.sub.1] receptor-mediated ANG II internalization and endogenous ANG II generation. The objective of the present study was to determine the relative contribution of de novo formation of endogenous ANG II. Male Sprague-Dawley rats were divided into three groups: sham operated (n = 6), [Val.sup.5]-ANG II infused (n = 16), and [Ile.sup.5]-ANG II infused (n = 6). [Val.sup.5]-ANG II and [Ile.sup.5]-ANG II were infused at 80 ng/min via subcutaneous osmotic minipump for 13 days, followed by harvesting of blood and kidney samples. In six [Val.sup.5]-ANG II-infused rats, urine was collected on the day before infusion and on day 12 of infusion. Extracted samples were subjected to HPLC to separate [Val.sup.5]-ANG II from [Ile.sup.5]-ANG II followed by RIA. Systolic blood pressure increased significantly from 121 [+ or -] 2 to 206 [+ or -] 4 mmHg in the [Val.sup.5]-ANG II-infused rats and from 124 [+ or -] 3 to 215 [+ or -] 5 mmHg in the [Ile.sup.5]-ANG II-infused rats. In the [Val.sup.5]-ANG II-infused rats, the plasma [Ile.sup.5]-ANG II levels increased 196.2 [+ or -] 70.1% compared with sham plasma [Ile.sup.5]-ANG II concentration. [Val.sup.5]-ANG II levels were 150.0 [+ or -] 28.2 fmol/ml which accounted for 53.5 [+ or -] 10.1% of the total ANG II in plasma. The kidney [Ile.sup.5]-ANG II levels in the [Val.sup.5]-ANG II-infused rats increased 69.9 [+ or -] 30.7% compared with sham kidney [Ile.sup.5]-ANG II concentrations. Intrarenal accumulation of [Val.sup.5]-ANG II accounted for 52.5 [+ or -] 5.3% of the total kidney ANG II during [Val.sup.5]-ANG II infusion while endogenous [Ile.sup.5]-ANG II accounted for 47.5 [+ or -] 8.6%. The urinary [Ile.sup.5]-ANG II excretion rate on day 12 increased 93.2 [+ or -] 32.1% compared with preinfusion level indicating increased formation of endogenous ANG II. Thus, the increases in intrarenal ANG II levels during chronic ANG II infusions involve substantial stimulation of endogenous ANG II formation which contributes to overall augmentation of intrarenal ANG II. angiotensin II-induced hypertension; renin-angiotensin system; high-performance liquid chromatography

We have developed a new method for contrast microangiography of the rat renal circulation using synchrotron radiation. The method was applied to determine responses of the renal arterial vasculature to angiotensin II and electrical stimulation of the renal nerves (RNS). Iodinated contrast agent was administered directly into the renal artery of pentobarbital-anesthetized rats before and during 1) intravenous infusion of angiotensin II (1.6 [micro]g x [kg.sup.-1] x [min.sup.-1]) or 2) its vehicle, or 3) RNS at 2 Hz. Images were obtained at 30 Hz, before and during these treatments, and vascular caliber was determined by use of a newly developed algorithm described herein. Up to four levels of branching could be observed simultaneously along the arterial tree, comprising vessels with resting diameter of 28-400 [micro]m. Vessel diameter was not significantly altered by vehicle infusion (+3.1 [+ or -] 3.5% change) but was significantly reduced by angiotensin II (-24.3 [+ or -] 3.4%) and RNS (- 17.1 [+ or -] 3.8%). Angiotensin H-induced vasoconstriction was independent of vessel size, but RNS-induced vasoconstriction was greatest in vessels with a resting caliber of 100-200 [micro]m and least in vessels with a resting caliber 40-100 [micro]m. In conclusion, the method we describe herein provides a new approach for assessing responses of the renal arterial circulation to vasoactive factors along several orders of branching. angiography; angiotensin II; vascular caliber; kidney circulation; renal nerves

It has been reported that mechanical strain activates extracellular signal-regulated protein kinases (ERK) without the involvement of angiotensin II (Ang II) in cardiomyocytes. We examined the effects of mechanical strain on ERK phosphorylation levels in the absence of Ang II using rat mesangial cells. The ratio of phosphorylated ERK (p-ERK) to total ERK expression was increased by cyclic mechanical strain in a timeand elongation strength-dependent manner. With olmesartan [Ang II type 1 receptor (AT1R) antagonist] pretreatment, p-ERK plateau levels decreased in a dose-dependent manner (E[C.sub.50] = 1.3 x [10.sup.-8] M, maximal inhibition 50.6 [+ or -] 11.0% at [10.sup.-5] M); a similar effect was observed with RNA interference against Ang II type I A receptor (A[T.sub.1A]R) and Tempol, a superoxide dismutase mimetic. In addition to the inhibition of p-ERK levels, olmesartan blocked the increase in cell surface and phosphorylated [p47.sup.phox] induced by mechanical strain and also lowered the mRNA expression levels of NADPH oxidase subunits. These results demonstrate that mechanical strain stimulates AT1R to phosphorylate ERK in mesangial cells in the absence of Ang II. This mechanotransduction mechanism is involved in the oxidative stress caused by NADPH oxidase and is blocked by olmesartan. The inverse agonistic activity of this AT1R blocker may be useful for the prevention of mesangial proliferation and renal damage caused by mechanical strain/oxidative stress regardless of circulating or tissue Ang II levels. hypertension

ANG II [AT.sub.1] receptor blockade reduces inflammation in hypertension. To determine whether ANG II [AT.sub.1] receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 [micro]g/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-[alpha], IL-1[beta], and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-[alpha], IL-1[beta], and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of [AT.sub.1] receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked [AT.sub.1] receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-[alpha] and IL-6 mRNA and protein; expression of IL-1[beta] and I[kappa]B-[alpha] mRNA; COX-2 mRNA and protein expression and PG[E.sub.2] concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that [AT.sub.1] receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions. CD14; cyclooxygenase-2; NAPDH oxidase; inducible nitric oxide synthase; proinflammatory cytokines

To better understand the patho-physiological role of angiotensin II (ANG II) in the dynamic autonomic regulation of heart rate (HR), we examined the effects of intravenous administration of ANG II (10 [micro]g x [kg.sup.-1] x [h.sup.-1]) on the transfer function from vagal or sympathetic nerve stimulation to HR in anesthetized rabbits with sinoaortic denervation and vagotomy. In the vagal stimulation group (n = 7), we stimulated the right vagal nerve for 10 min using binary white noise (0-10 Hz). The transfer function from vagal stimulation to HR approximated a first-order low-pass filter with pure delay. ANG II attenuated the dynamic gain from 7.6 [+ or -] 0.9 to 5.8 [+ or -] 0.9 beats x [min.sup.-1] x [Hz.sup.-1] (means [+ or -] SD; P < 0.01) without affecting the corner frequency or pure delay. In the sympathetic stimulation group (n = 7), we stimulated the right postganglionic cardiac sympathetic nerve for 20 min using binary white noise (0-5 Hz). The transfer function from sympathetic stimulation to HR approximated a second-order low-pass filter with pure delay. ANG II slightly attenuated the dynamic gain from 10.8 [+ or -] 2.6 to 10.2 [+ or -] 3.1 beats x [min.sup.-1] x [Hz.sup.-l] (p = 0.049) without affecting the natural frequency, damping ratio, or pure delay. The disproportional suppression of the dynamic vagal and sympathetic regulation of HR would result in a relative sympathetic predominance in the presence of ANG II. The reduced high-frequency component of HR variability in patients with cardiovascular diseases, such as myocardial infarction and heart failure, may be explained in part by the peripheral effects of ANG II on the dynamic autonomic regulation of HR. systems analysis; transfer function; heart rate variability; cardiac sympathetic nerve activity; rabbit

Infusion of ANG II in hyperlipidemic mice augments atherosclerosis and causes formation of abdominal aortic aneurysms (AAAs). The purpose of this study was to define the contribution of ANG II-induced hypertension to these vascular pathologies. Male apolipoprotein E (apoE)- and LDL receptor (LDLr)-deficient mice were infused with ANG II (1,000 ng x [kg.sup.-1] x [min.sup.-1]) or norepinephrine (NE; 5.6 mg x [kg.sup.-1] x [day.sup.-1]) for 28 days. Infusion of ANG II or NE increased mean arterial pressure (MAP; ANG II, 133 [+ or -] 2.8; NE, 129 [+ or -] 13 mmHg) to a similar extent compared with baseline blood pressures (MAP, 107 [+ or -] 2 mmHg). Abdominal aortic width increased in both apoE-deficient (apo[E.sup.-/-]) or LDLr-deficient (LD[Lr.sup.-/-]) mice infused with ANG II (apo[E.sup.-/-]: 1.4 [+ or -] 0.1; LD[Lr.sup.-/-]: 1.6 [+ or -] 0.2 mm). In contrast, NE did not change diameters of abdominal aortas (apo[E.sup.-/-]: 0.91 [+ or -] 0.03; LD[Lr.sup.-/-]: 0.87 [+ or -] 0.02 mm). Similarly, atherosclerotic lesions in aortic arches were much greater in mice infused with ANG II compared with NE. At a subpressor infusion rate of ANG II (500 ng x [kg.sup.-1] x [min.sup.-1]), AAAs developed in 50% of apo[E.sup.-/-] mice. Alternatively, administration of hydralazine (250 mg/l) to ANG II-infused apo[E.sup.-/-] mice (1,000 ng x [kg.sup.-1] x [min.sup.-1]) lowered systolic blood pressure (day 28: ANG II, 157 [+ or -] 6; ANG II/hydralazine, 135 [+ or -] 6 mmHg) but did not prevent AAA formation or atherosclerosis. These results demonstrate that infusion of ANG II to hyperlipidemic mice induces AAAs and augments atherosclerosis independent of increased blood pressure. aneurysms; hypertension; vascular lesions

ANG II type 1 receptors ([AT.sub.1]R) mediate most of the central effects of ANG II on cardiovascular function, fluid homeostasis, and sympathetic drive. The mechanisms regulating [AT.sub.1]R expression in the brain are unknown. In some tissues, the [AT.sub.1]R can be upregulated by prolonged exposure to ANG II. We examined the hypothesis that ANG II upregulates the [AT.sub.1]R in the brain by stimulating the intracellular mitogen-activated protein kinase (MAPK) signaling pathway. Using molecular and immunochemical approaches, we examined expression of the [AT.sub.1]R and phosphorylated MAPK in the paraventricular nucleus of the hypothalamus (PVN) and the subfornical organ (SFO) of rats receiving a chronic (4-wk) subcutaneous infusion of ANG II (0.6 [micro]g/h) or saline (vehicle control), with or without concomitant (4-wk) intracerebroventricular (ICV) infusions of MAPK inhibitors or the [AT.sub.1]R blocker losartan. Subcutaneous infusion of ANG II markedly increased phosphorylation of MAPK and expression of [AT.sub.1]R mRNA and protein and [AT.sub.1]R-like immunoreactivity in the PVN and SFO. ANG II-induced [AT.sub.1]R expression was blocked by ICV infusion of the p44142 MAPK inhibitor PD-98059 (0.025 [micro]g/h) and the JNK inhibitor SP-600125 (0.125 [micro]g/h), but not by the p38 MAPK inhibitor SB203580 (0.125 [micro]g/h). Upregulation of the [AT.sub.1]R in the PVN and SFO by peripheral ANG II was abolished by ICV losartan (10 [micro]g/h). The data indicate that blood-borne ANG II upregulates brain [AT.sub.1]R by activating intracellular p44/42 MAPK and JNK signaling pathways. paraventricular nucleus; subfornical organ; angiotensin II type 1 receptor; autonomic regulation

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3-/- mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3-/- mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3-/- mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased ATIR expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression. angiotensin II type 1 receptor; acetylcholine receptor; CXCR3 knock-out mice; EMSA; transcription factors; vascular contractility

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 [+ or -] 24 fmol/mg compared with 144 [+ or -] 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 [+ or -] 15 and 62 [+ or -] 7 fmol/mg, respectively, in anephric rats compared with 172 [+ or -] 8 fmol/mg for ANG I and 42 [+ or -] 4 fmol/mg for ANG II in controls (P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and [AT.sub.1] receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in [p47.sup.phox], Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin. renin; angiotensinogen; angiotensin II; blood pressure

The early stage of age-related macular degeneration (AMD) is characterized by the formation of subretinal pigment epithelium (RPE) deposits as a result of the dysregulation in the turnover of extracellular matrix (ECM) molecules. However, the mechanism involved remains unclear. Hypertension (HTN) is an important risk factor for AMD, and angiotensin II (ANG II) is the most important hormone associated with HTN. However, the relevance of ANG II receptors and ANG II effects on RPE have not been investigated yet. Therefore, the expression and regulation of ANG II receptors as well as the ECM turnover were studied in human RPE. ANG II receptors were expressed and upregulated by ANG II in human RPE. This regulation resulted in functional receptor expression, since an increase in intracellular concentration of calcium was observed upon ANG II stimulation. ANG II also increased matrix metalloproteinase (MMP)-2 activity and MMP-14 at the mRNA and protein levels as well as type IV collagen degradation. These ANG II effects were abolished in the presence of the ANG II receptor subtype 1 (AT1) receptor antagonist candesartan. In contrast, ANG II decreased type IV collagen via both AT1 and AT2 receptors, suggesting a synergistic effect of the two receptor subtypes. In conclusion, we have confirmed the presence of ANG II receptors in human RPE and their regulation by ANG II as well as the regulation of ECM molecules via ANG II receptors. Our data support the hypothesis that ANG II may exert biological function in RPE through ANG II receptors and that ANG II may cause dysregulation of molecules that play a major role in the turnover of ECM in RPE basement membrane and Bruch's membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD. calcium; hypertension; Bruch's membrane

Augmented intrarenal ANG II stimulates 1L-6, which contributes to renal injury. The expression of intrarenal angiotensinogen (AGT) is enhanced by increased intrarenal ANG II in human renin/human AGT double transgenic mice. ANG II also augments AGT expression in hepatocytes and cardiac myocytes. However, the mechanisms underlying AGT augmentation by ANG II and the contribution of IL-6 to this system are poorly understood. This study was performed in human renal proximal tubular epithelial cells (HRPTECs) to test the hypothesis that IL-6 contributes to the upregulation of AGT expression by ANG II. Human kidney-2 (HK-2) cells, immortalized HRPTECs, were incubated with [10.sup.-7] M ANG II and/or 10 ng/ml IL-6 for up to 24 h. AGT mRNA and protein expressions were measured by real-time RT-PCR and ELISA, respectively. The activities of NF-[kappa]B and STAT3 were evaluated by Western blotting and EMSA. Stimulation with either ANG II or IL-6 did not significantly alter AGT mRNA or protein expression. In contrast, costimulation with ANG II and IL-6 significantly increased AGT mRNA and protein expressions (1.26 [+ or -] 0.10 and 1.16 [+ or -] 0.13 over control, respectively). Olmesartan, an ANG II type 1 receptor blocker, and an IL-6 receptor antibody individually inhibited this synergistic effect. NF-[kappa]B was also activated by costimulation with ANG II and 1L-6. Phosphorylation and activity of STAT3 were increased by stimulation with IL-6 alone and by costimulation. The present study indicates that IL-6 plays an important role in ANG II-mediated augmentation of AGT expression in human renal proximal tubular cells. angiotensinogen; kidney; renin-angiotensin system; cytokines

The brain renin-angiotensin system plays an important role in the regulation of arterial pressure in response to stress, in part due to activation of A[T.sub.1] receptors in the hypothalamic median preoptic nucleus (MnPO) by endogenous angiotensin II (ANG II). N-methyl-D-aspartate (NMDA) receptors are also involved in the angiotensinergic signaling pathway through the MnPO. We investigated whether A[T.sub.1] and NMDA receptors in the MnPO are responsible for variable hemodynamic response patterns to stress. Cocaine or startle with cold water evoked a pressor response in Sprague-Dawley rats due, in some rats [vascular responders (VR)], to a large increase in systemic vascular resistance (SVR) and, in other rats [mixed responders (MR)], to small increases in SVR and cardiac output (CO). Microinjection of the GABAA agonist muscimol into the MnPO to block synaptic transmission attenuated the cocaine-or stress-induced increase in SVR and the decrease in CO seen in VR without altering either response in MR. Likewise, administration of either an A[T.sub.1] receptor antagonist, losartan, or an NMDA receptor antagonist, MK-801, attenuated the increase in SVR and the decrease in CO in VR in response to either cocaine (losartan and MK-801) or startle with cold water (losartan) without altering either response in MR. We propose that the MnPO is responsible for greater SVR responses in VR and that A[T.sub.1] and NMDA receptors play an important role in greater SVR responses in VR. These data provide additional support for the critical role of the MnPO in cardiovascular responses to stress. A[T.sub.1] receptor; cocaine; anteroventral third ventricle region; systemic vascular resistance; cardiovascular response variability

Diabetes and insulin resistance are associated with an increased risk of hypertension and cardiovascular disease. Recent evidence demonstrates that A[T.sub.2] receptors (AT2R) play an important role in the hemodynamic control of hypertension by vasodilation. The quantitative significance of AT2R in the establishment of diabetic vascular dysfunction, however, is not well defined and needs further investigation. Goto-Kakizaki (GK) rats, a polygenic model of spontaneous normotensive type 2 diabetes, were used to examine any abnormalities in cardiovascular function associated with AT2R at the early stage of the disease without endothelium influence. Using a myograph to measure the isometric force, we observed that ANG II-induced contraction was impaired in denuded GK aorta compared with control Wistar-Kyoto (WKY) aorta and exhibited a retarded AT1R antagonist response and enhanced Rho kinase signaling. When AT1R were blocked, ANG II induced a significant vasodilation of precontracted GK aorta via AT2R. The protein and mRNA of AT2R were increased in diabetic GK denuded aorta. Blocking AT2R restored the ANG II-induced contraction in the GK vasculature to control levels, demonstrating a counteractive role for AT2R in AT1R-induced contraction. Inhibition of inducible nitric oxide synthase (iNOS) by [N.sup.G]-monomethyl-L-arginine mimicked AT2R inhibition in denuded GK aorta, suggesting that AT2R-induced vasodilation was dependent on iNOS/NO generation. The protein and mRNA of iNOS were also increased in GK aorta. In conclusion, these results clearly demonstrate that enhanced AT2R and iNOS-induced, NO-mediated vasodilation impair ANG II-induced contraction in an endothelium-independent manner at the early stage of type 2 diabetes. angiotensin type 2 receptor; inducible nitric oxide synthase; vasodilation

Angiotensin II (Ang II) stimulation of the Ang type 1 receptor (A[T.sub.I]R) facilitates myocardial remodeling through NADPH oxidase-mediated generation of oxidative stress. Components of the renin-angiotensin system constitute an autocrine/paracrine unit in the myocardium, including renin, which is the rate-limiting step in the generation of Ang II. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo renin inhibition and/or A[T.sub.1]R blockade in a rodent model of chronically elevated tissue Ang II levels, the transgenic (mRen2)27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, and cardiovascular damage. Young (6- to 7-wk-old) heterozygous (+/-) male Ren2 and age-matched Sprague-Dawley rats were treated with the renin inhibitor aliskiren, which has high preferential affinity for human and mouse renin, an A[T.sub.1]R blocker, irbesartan, or placebo for 3 wk. Myocardial NADPH oxidase activity and immunostaining for NADPH oxidase subunits and 3-nitrotyrosine were evaluated and remodeling changes assessed by light and transmission electron microscopy. Blood pressure, myocardial NADPH oxidase activity and subunit immunostaining, 3-nitrotyrosine, perivascular fibrosis, mitochondrial content, and markers of activity were significantly increased in Ren2 compared with SD littermates. Both renin inhibition and blockade of the A[T.sub.1]R significantly attenuated cardiac functional and structural alterations, although irbesartan treatment resulted in greater reductions of both blood pressure and markers of oxidative stress. Collectively, these data suggest that both reduce changes driven, in part, by Ang II-mediated increases in NADPH oxidase and, in part, increases in blood pressure. cardiac remodeling; reduced nicotinamide adenine dinucleotide phosphate oxidase; oxidative stress

[Ca.sup.+] influx across the plasma membrane is a major component of mesangial cell (MC) response to vasoconstrictors. Polycystin 2 (PC2), the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as a nonselective cation channel in a variety of cell types. The present study was performed to test the hypothesis that PC2 and its binding partners constitute a [Ca.sup.2+]-permeable channel and contribute to ANG II-induced [Ca.sup.2+] signaling in MCs. Western blot and immunocy-to-chemistry showed PC2 expression in cultured human MCs. The existence of PC2 in MCs was further confirmed by immunohistochemsitry in rat kidney sections. Coimmunoprecipitation displayed a selective interaction of PC2 with canonical transient receptor potential (TRPC) proteins TRPC1 and TRPC4. Cell-attached patch-clamp experiments revealed that ANG II-induced membrane currents were enhanced by over-expression of pkd2 but significantly inhibited by knock down of pkd2, 30 [micro]M [Gd.sup.3+] (a PC2 channel blocker), and dominant-negative pkd2 mutant (pkd2-D511V). Corresponding to the increase in channel currents, ANG II stimulation increased expression of PC2 on the cell surface of MCs and interaction with TRPC1 and TRPC4. Furthermore, ANG H-induced MC contraction was significantly reduced in pkd2-knocked down MCs. These data suggest that PC2 selectively assembles with TRPC1 and TRPC4 to form channel complexes mediating ANG II-induced [Ca.sup.2+] responses in MCs. calcium channel

ADP-ribosyl cyclase (ADPR-cyclase) produces a [Ca.sup.2+]-mobilizing second messenger cyclic ADP-ribose (cADPR) from NA[D.sup.+]. In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular [Ca.sup.2+] concentrations through a transient [Ca.sup.2+] release via an inositol 1,4,5-trisphosphate receptor and a sustained [Ca.sup.2+] influx via L-type [Ca.sup.2+] channels. The sustained [Ca.sup.2+] signal, but not the transient [Ca.sup.2+] signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPRcyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-[gamma]1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [[sup.3]H]thymidine and [[sup.3]H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis. cyclic ADP-ribose; intracellular [Ca.sup.2+] concentration