II-induced Hypertensive Rats Research Articles

SARS-CoV-2 Spike Protein S1 Exposure Increases Susceptibility to Angiotensin II-Induced Hypertension in Rats by Promoting Central Neuroinflammation and Oxidative Stress.

The SARS-CoV-2 spike S1 subunit (S1) can cross the blood-brain barrier and elicit neuroinflammatory response independent of viral infection. Here we examined whether S1 influences blood pressure (BP) and sensitizes the hypertensive response to angiotensin (ANG) II by enhancing neuroinflammation and oxidative stress in hypothalamic paraventricular nucleus (PVN), a key brain cardiovascular regulatory center. Rats received central S1 or vehicle (VEH) injection for 5days. One week after injection, ANG II or saline (control) was subcutaneously delivered for 2weeks. S1 injection induced greater increases in BP, PVN neuronal excitation and sympathetic drive in ANG II rats but had no effects in control rats. One week after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker were higher but mRNA of Nrf2, the master regulator of inducible antioxidant and anti-inflammatory responses, was lower in the PVN in S1-injected rats than in VEH-injected rats. Three weeks after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker, microglia activation and reactive oxygen species in the PVN were comparable between S1 and VEH treated control rats but were elevated in two groups of ANG II rats. Notably, ANG II-induced elevations in these parameters were exaggerated by S1. Interestingly, ANG II increased PVN Nrf2 mRNA in VEH-treated rats but not in S1-treated rats. These data suggest that S1 exposure has no effect on BP, but post-S1 exposure increases susceptibility to ANG II-induced hypertension by downregulating PVN Nrf2 to promote neuroinflammation and oxidative stress and augment sympathetic excitation.

Activation of the Mas receptors by AVE0991 and MrgD receptor using alamandine to limit the deleterious effects of Ang II-induced hypertension.

The MrgD receptor agonist, alamandine (ALA) and Mas receptor agonist, AVE0991 have recently been identified as protective components of the renin-angiotensin system. We evaluated the effects of ALA and AVE0991 on cardiovascular function and remodeling in angiotensin (Ang) II-induced hypertension in rats. Sprague Dawley rats were subject to 4-week subcutaneous infusions of Ang II (80 ng/kg/min) or saline after which they were treated with ALA (50 μg/kg), AVE0991 (576 μg/kg), or ALA+AVE0991 during the last 2 weeks. Systolic blood pressure (SBP) and heart rate (HR) values were recorded with tail-cuff plethysmography at 1, 15, and 29 days post-treatment. After euthanization, the heart and thoracic aorta were removed for further analysis and vascular responses. SBP significantly increased in the Ang II group when compared to the control group. Furthermore, Ang II also caused an increase in cardiac and aortic cyclophilin-A (CYP-A), monocyte chemoattractant protein-1 (MCP-1), and cardiomyocyte degeneration but produced a decrease in vascular relaxation. HR, matrix metalloproteinase-2 and -9, NADPH oxidase-4, and lysyl oxidase levels were comparable among groups. ALA, AVE0991, and the drug combination produced antihypertensive effects and alleviated vascular responses. The inflammatory and oxidative stress related to cardiac MCP-1 and CYP-A levels decreased in the Ang II+ALA+AVE0991 group. Vascular but not cardiac angiotensin-converting enzyme-2 levels decreased with Ang II administration but were similar to the Ang II+ALA+AVE0991 group. Our experimental data showed the combination of ALA and AVE0991 was found beneficial in Ang II-induced hypertension in rats by reducing SBP, oxidative stress, inflammation, and improving vascular responses.

Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin II-induced hypertension in rats.

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine 105 (S105) for the actions of GATA4 in an angiotensin II (AngII)‐induced hypertension rat model. Adenoviral constructs overexpressing wild‐type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg/kg/h) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT‐qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII‐induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult‐to‐foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild‐type or mutated GATA4. Our results indicate that GATA4 reduces AngII‐induced responses by interfering with pro‐fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.

Angiotensin II-induced hypertension in rats is only transiently accompanied by lower renal oxygenation

Activation of the renin-angiotensin system may initiate chronic kidney disease. We hypothesised that renal hypoxia is a consequence of hemodynamic changes induced by angiotensin II and occurs prior to development of severe renal damage. Male Sprague-Dawley rats were infused continuously with angiotensin II (350 ng/kg/min) for 8 days. Mean arterial pressure (n = 5), cortical (n = 6) and medullary (n = 7) oxygenation (pO2) were continuously recorded by telemetry and renal tissue injury was scored. Angiotensin II increased arterial pressure gradually to 150 ± 18 mmHg. This was associated with transient reduction of oxygen levels in renal cortex (by 18 ± 2%) and medulla (by 17 ± 6%) at 10 ± 2 and 6 ± 1 hours, respectively after starting infusion. Thereafter oxygen levels normalised to pre-infusion levels and were maintained during the remainder of the infusion period. In rats receiving angiotensin II, adding losartan to drinking water (300 mg/L) only induced transient increase in renal oxygenation, despite normalisation of arterial pressure. In rats, renal hypoxia is only a transient phenomenon during initiation of angiotensin II-induced hypertension.

The standardized extract of Nigella sativa and its major ingredient, thymoquinone, ameliorates angiotensin II-induced hypertension in rats.

Background This study investigated the effect of hydroalcoholic extract of Nigella sativa (N. sativa) and its active component, thymoquinone (TQ) on hypertension induced by angiotensin II (AngII), the main product of renin-angiotensin system (RAS). Methods Seven animal groups (n=7 for each group) were used as follows: (1) control, (2) AngII (300 ng/kg), (3) AngII+losartan (Los; 10 mg/kg), (4) TQ (40 mg/kg)+AngII, and (5-7) three doses of N. sativa (200, 400, and 600 mg/kg)+AngII. Los and AngII were injected intravenously; TQ and extracts were injected intraperitoneally. In TQ and N. sativa-treated groups, 30 min after injection of the extract and TQ, AngII was injected. Cardiovascular parameters were recorded by power lab system after cannulation of femoral artery. The maximum changes (∆) of systolic blood pressure (SBP), mean arterial pressure (MAP), and heart rate (HR) were calculated and used for statistical analysis. Results AngII significantly increased maximal ∆SBP, ∆MAP, and ∆HR compared with the control (p<0.001), and these effects significantly were blunted by Los. TQ and two higher doses (400 and 600 mg/kg) of N. sativa significantly could antagonize effect of AngII on ∆SBP, ∆MAP (p<0.05 to p<0.001). AngII-induced changes of HR are also significantly decreased by TQ and dose 600 mg/kg of extract (p<0.01 and p<0.05, respectively). Conclusions The N. sativa and its component TQ have the beneficial effect on hypertension probably due to attenuation cardiovascular effects of AngII.

Intrarenal ghrelin receptor inhibition ameliorates angiotensin II-dependent hypertension in rats.

The intrarenal ghrelin receptor (GR) is localized to collecting duct (CD) cells, where it increases epithelial Na+ channel (αENaC)-dependent sodium reabsorption in rodents. We hypothesized that chronic GR inhibition with intrarenal GR siRNA lowers blood pressure (BP) in angiotensin II-dependent hypertension via reductions in αENaC-dependent sodium reabsorption. Uninephrectomized Sprague-Dawley rats ( n = 121) received subcutaneous osmotic pumps for chronic systemic delivery of angiotensin II or vehicle (5% dextrose in water). Rats also received intrarenal infusion of vehicle, GR siRNA, or scrambled (SCR) siRNA. In rats receiving intrarenal vehicle or intrarenal SCR siRNA, systemic angiotensin II infusion increased sodium retention and BP on day 1, and BP remained elevated throughout the 5-day study. These rats also demonstrated increased CD GR expression after 5 days of infusion. However, intrarenal GR siRNA infusion prevented angiotensin II-mediated sodium retention on day 1, induced a continuously negative cumulative sodium balance compared with angiotensin II alone, and reduced BP chronically. Glomerular filtration rate and renal blood flow remained unchanged in GR siRNA-infused rats. Systemic angiotensin II infusion also increased serum aldosterone levels, CD αENaC, and phosphorylated serum and glucocorticoid-inducible kinase 1 expression in rats with intrarenal SCR siRNA; however, these effects were not observed in the presence of intrarenal GR siRNA, despite exposure to the same systemic angiotensin II. These data demonstrate that chronic inhibition of intrarenal GR activity significantly reduces αENaC-dependent sodium retention, resulting in a negative cumulative sodium balance, thereby ameliorating angiotensin II-induced hypertension in rats. Renal GRs represent a novel therapeutic target for the treatment of hypertension and other sodium-retaining states.

Abstract P436: Intrarenal Ghrelin Receptor Inhibition Prevents Sodium Retention and Reduces Blood Pressure in Angiotensin II-induced Hypertension

The renal ghrelin receptor (GR) is localized to collecting duct (CD) cells where it increases E Na C-dependent sodium (Na + ) reabsorption. We have previously shown that in uninephrectomized Sprague-Dawley (SD) rats, systemic Ang II infusion induces hypertension after 1 day (129.2 ± 5 vs. 158.2 ± 2.6 mmHg; P&lt;0.01), and this increase is sustained over a 6 day period (191.2 ± 4.0 mmHg; P&lt;0.001). The increase in BP is attenuated with concomitant intrarenal (IR) GR siRNA infusion (F=28.9; P&lt;0.001), but the mechanism is unknown. In this study, we tested whether the reduction in Ang II-induced hypertension in the presence of IR GR siRNA is the result of the prevention of antinatriuresis. Uninephrectomized SD rats (N=33) received a subcutaneous osmotic pump for chronic systemic delivery of Ang II (200 ng/kg/min) or 5% dextrose in water (D 5 W). Rats received IR bolus infusion of D 5 W, GR siRNA (15 μg x 3 over 6 days), or scrambled siRNA (SCR siRNA, 15 μg x 3 over 6 days). Systemic Ang II + SCR siRNA reduced 24 h U Na V from 0.90 ± 0.04 to 0.34 ± 0.08 μmol/min (P&lt;0.001) and increased cumulative Na + balance from 0 to 0.54 mEq Na + (P&lt;0.01) on day 1, with no significant differences for either group for the remainder of the study. IR GR siRNA infusion prevented Ang II-mediated Na + retention (0.86 ± 0.06 vs. 0.87 ± 0.06 μmol/min) and induced a decrease in cumulative Na + balance from 0 to -0.37 ± 0.08 mEq on day 1 with no significant differences for either group for the remainder of the study. At 24 h of Ang II + SCR siRNA infusion, IR GR expression was increased in whole cell kidney (0.55 ± 0.03 vs. 0.91 ± 0.08 DU; P&lt;0.05) and major CD Na + transporter E Na C translocated from intracellular sites to apical plasma membranes to mediate Na + reabsorption (0.34 ± 0.05 vs. 0.51 ± 0.02 DU; P&lt;0.05). In rats receiving IR GR siRNA infusion, however, Ang II failed to recruit E Na C to the apical plasma membranes of CD cells. There was no change in total whole cell E Na C for all conditions. These data demonstrate that chronic inhibition of IR GR activity significantly reduces E Na C-dependent Na + retention, resulting in a negative cumulative Na + balance, thereby preventing Ang II-induced hypertension in rats. Thus, renal GRs represent a novel therapeutic target for the treatment of hypertension and other Na + -retaining states.

Renal medullary (pro)renin receptor contributes to angiotensin II-induced hypertension in rats via activation of the local renin-angiotensin system.

Background(Pro)renin receptor (PRR) is a new component of the renin–angiotensin system and regulates renin activity in vitro. Within the kidney, PRR is highly expressed in the renal medulla where its expression is induced by angiotensin II infusion. The objective of the present study was to test a potential role of renal medullary PRR during angiotensin II-induced hypertension.MethodsA rat AngII infusion model (100 ng/kg/min) combined with renal intramedullary infusion of PRO20, a specific inhibitor of PRR, was builded. And the intravenous PRO20 infusion serve as control. Mean arterial pressure was recorded by radiotelemetry for one week. Further anaylsis of kidney injury, inflammation, biochemical indices and protein localization were perrformed in vivo or in vitro.ResultsRadiotelemetry demonstrated that AngII infusion elevated the mean arteria pressure from 108 ± 5.8 to 164.7 ± 6.2 mmHg. Mean arterial pressure decreased to 128.6 ± 5.8 mmHg (P < 0.05) after intramedullary infusion of PRO20, but was only modestly affected by intravenous PRO20 infusion. Indices of kidney injury, including proteinuria, glomerulosclerosis, and interstitial fibrosis, inflammation, and increased renal medullary and urinary renin activity following angiotensin II infusion were all remarkably attenuated by intramedullary PRO20 infusion. Following one week of angiotensin II infusion, increased PRR immunoreactivity was found in vascular smooth muscle cells. In cultured rat vascular smooth muscle cells, angiotensin II induced parallel increases in soluble PRR and renin activity, and the latter was significantly reduced by PRO20.ConclusionRenal medullary PRR mediates angiotensin II-induced hypertension, likely by amplifying the local renin response.

Abstract MP05: Mineralocorticoid And Angiotensin II Type 1 Receptors In The Subfornical Organ Mediate Angiotensin II Hypertension In Rats

Circulating Ang II increases BP through both peripheral and central mechanisms. Activation of both AT 1 R and MR in the CNS plays a critical role in Ang II-induced hypertension. The subfornical organ (SFO) contains both MR and AT 1 R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN. In this study, we evaluated the effect of intra-SFO infusion of AAV-MR-siRNA or AAV-AT 1a R-siRNA on MR or AT 1 R mRNA expression in the SFO and PVN and Ang II induced hypertension as assessed by telemetry. Two week sc infusion of Ang II at 500 ng/kg/min in Wistar rats on regular salt diet increased BP by 60~65 mmHg. This pressor effect of Ang II was largely prevented by AAV-MR-siRNA or AAV-AT 1 R-siRNA in the SFO (Figure). Ang II increased AT 1 R mRNA expression (1.7± 0.4 vs 0.8 ± 0.04 х10 -1 , p&lt;0.05) in the SFO. Both MR- and AT 1 R-siRNA in the SFO prevented this increase (1.1 ± 0.2х10 -1 and 1.0± 0.3х10 -1 ). In contrast, Ang II decreased MR mRNA expression (3.2 ± 0.2 vs. 4.1 ± 0.2х10 -2 , p&lt;0.05) in the SFO. Both MR- and AT 1 R-siRNA further decreased MR mRNA expression (2.7 ± 0.2 х10 -2 and 2.3 ± 0.3 х10 -2 ). In the PVN, Ang II increased AT 1 R mRNA by ~100% and MR mRNA by ~20%. Only AT 1 R-siRNA in the SFO prevented these increases in the PVN. AAV mediated eGFP fluorescence expression was observed in the SFO but not the PVN. These results suggest that circulating Ang II differentially regulates MR and AT 1 R expression in the SFO. Prevention of Ang II induced AT 1 R up-regulation by knockdown of either AT 1 R or MR suggests a MR dependent regulation of AT 1 R in the SFO. MR-AT 1 R signaling in the SFO appears to play a major role in Ang II-induced hypertension in rats. * p&lt;0.05, vs. baseline values; a: p&lt;0.05, vs. AAV-SCM-siRNA

Knockdown of mineralocorticoid or angiotensin II type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin II hypertension in rats.

Circulating Ang II activates an aldosterone-mineralocorticoid receptor (MR) - angiotensin II (Ang II) - angiotensin type 1 receptor (AT1R) pathway in the hypothalamus. To obtain insights into the actual neuronal projections involved, adeno-associated virus carrying small interfering RNA against either AT1aR (AAV-AT1aR-siRNA) or MR (AAV-MR-siRNA) were infused into the paraventricular nucleus (PVN) in Wistar rats. Intra-PVN infusion of AAV-AT1aR-siRNA or AAV-MR-siRNA decreased AT1R or MR expression in the PVN but not in the subfornical organ (SFO) or supraoptic nucleus (SON). Subcutaneous infusion of Ang II at 500ngkg(-1)min(-1) for 2weeks increased mean arterial pressure by 60-70mmHg, and increased AT1R and MR expression in the SFO, SON and PVN. Intra-PVN AT1aR-siRNA prevented the Ang II-induced increase in AT1R but not MR expression in the PVN, and MR-siRNA prevented MR but not AT1R expression in the PVN. The increases in AT1R and MR expression in both the SFO and the SON were not changed by the two AAV-siRNAs. Specific knockdown of AT1R or MR in the PVN by AAV-siRNA each prevented most of the Ang II-induced hypertension. Prevention of the subcutaneous Ang II-induced increase in MR but not the increase in AT1R by knockdown of MR and vice versa suggests an independent regulation of MR and AT1R expression in the PVN. Both AT1R and MR activation in the PVN play a critical role in Ang II-induced hypertension in rats.

Abstract 154: Knockdown of Mineralocorticoid or AT 1 Receptor Gene in the Paraventricular Nucleus Prevents Ang II Induced Hypertension in Rats

Circulating Ang II activates an aldosterone-MR-AT 1 R pathway in the hypothalamus. To obtain insights into the actual aldosterone-MR- AT 1 R neuronal pathway, the present study examined whether intra-PVN infusion of AAV-AT 1a R-siRNA or AAV-MR-siRNA prevents the Ang II induced increases in AT 1 R and MR expression and BP. Two weeks sc infusion of Ang II at 500ng/kg/min in Wistar rats on regular salt diet increased BP by telemetry by 66 ± 3 mm Hg, and AT 1 R and MR protein in the SFO, SON and PVN. Intra-PVN AT 1a R-siRNA decreased AT 1 R but not MR expression in the PVN by sc Ang II, and MR-siRNA decreased MR but not AT 1 R expression in the PVN. AT 1 R and MR expression in both SFO and SON were not significantly changed by the two AAV-siRNA. Specific knockdown of AT 1 R or MR in the PVN by AAV-siRNA prevented ~80% of the increase in BP induced by sc Ang II. These results suggest that circulating Ang II up-regulates brain Ang II and aldosterone and increases MR and AT 1 R in the SFO, SON and PVN. Prevention of Ang II induced increases in MR but not AT 1 R by knockdown of MR and vice versa suggests an independent regulation of MR and AT 1 R in the PVN. Both AT 1 R and MR in the PVN play a critical role in Ang II-induced hypertension in rats. * p&lt;0.05 versus AAV-SCM alone. a: p&lt;0.05 versus AAV-SCM + Ang II.

MR or AT1R gene silencing in the PVN markedly attenuates Ang II‐induced hypertension in rats

Functional studies suggest that circulating Ang II activates an aldosterone‐MR‐endogenous ouabain (EO)‐ AT1R pathway in the hypothalamus and thereby causes hypertension. The present study examined whether intra‐PVN infusion of AAV‐MR‐siRNA or AAV‐AT1aR‐siRNA attenuates Ang II‐induced hypertension in rats. Two wks after bilateral intra‐PVN infusion of AAV‐MR‐siRNA (1 μl of 2.5 × 1010 genomic particles / ml), MR mRNA and protein expression in the PVN were reduced by 56% and 31% (P&lt;0.01), compared with rats infused with AAV‐SCM‐siRNA. In 2nd set of rats, 1 week following bilateral intra‐PVN infusion of AAV‐MR‐siRNA, AAV‐AT1aR‐siRNA (1 μl of 2.5 × 1010 genomic particles / ml) or AAV‐SCM‐siRNA, Ang II was infused subcutaneously at 500ng/kg/min. After 2 weeks, AAV‐AT1aR‐siRNA significantly attenuated, whereas AAV‐MR‐siRNA largely prevented the Ang II‐induced hypertension. These results suggest that MR activation in the PVN plays a major role in Ang II‐induced hypertension, and genetic intervention of MR or AT1R production by AAV‐siRNA into specific nuclei provides new insights into the actual pathways of MR‐EO neuromodulation. MAP (mmHg) HR (bpm) AAV‐SCM‐siRNA ( n=5) 113 ± 2 377 ± 8 AAV‐SCM‐siRNA +Ang II ( n=5) 192 ± 7 a 416 ± 15 AAV‐AT1aR‐siRNA+ Ang II ( n=7) 153 ± 8*, Δ 411 ± 12 AAV‐MR‐siRNA + Ang II ( n=7) 121 ± 6 377 ± 12 Data=means± SE. p&lt;0.05, vs. others, p&lt;0.05, vs. AAV‐SCM, p&lt;0.05, vs. AAV‐MR. (Supported by grant # FRN:MOP‐74432 from the Canadian Institutes of Health Research)

Abstract 413: Electroacupuncture Improves Arterial Baroreflex and Chemoreflex Sensitivity in Angiotensin II-induced Hypertension in Rats

Despite the use of electroacupuncture (EA) to treat a number of cardiovascular diseases, little is known about the mechanisms that underlie its actions. It is well known that renin-angiotensin system plays a major role in pathogenesis of hypertension. The purpose of this study was to examine the influence of EA stimulation of ST 36 on arterial baro- and chemoreflex sensitivity in angiotensin (Ang) II-induced hypertension in rats. Four groups of SD rats (n=8/group) were implanted with subcutaneous osmotic minipumps for infusion of Ang II (300 ng/kg/min) or saline control, with or without EA stimulation. EA groups underwent a 2 Hz EA treatment at the acupoints ST36 or a non-acupoint with the intensity increasing stepwise from 0.5-1-1.5 mA once a day for 30 min. Baroreflex sensitivity index was calculated on the basis of reflex changes in heart rate (HR) related to the arterial pressure (AP) changes induced by phenylephrine or sodium nitroprusside injection, and chemoreflex sensitivity was evaluated by intravenous injections of increasing doses of potassium cyanide (KCN). After 4 weeks of treatment, Ang II significantly increased mean arterial pressure (107±2 and 139±2 mmHg in control vs. Ang II group). Baroreflex sensitivity to AP rises and falls were also impaired in Ang II infusion group (1.3±0.08 and 2.1 ±0.18 bpm/mmHg) compared with those in control group (1.7±0.08 and 2.7±0.06 bpm/mmHg). EA stimulation of ST 36 on Ang II infusion rats significantly improved the impaired baroreflex sensitivity (1.7±0.06 and 2.6±0.1 bpm/mmHg, P &lt; 0.05). In addition, the bradycardic and pressor responses evoked by KCN injections were attenuated in Ang II infusion rats (23±9 bpm and 7±1 mmHg in Ang II group vs. 78±7 bpm and 22±3mmHg in control group, P &lt; 0.05), while EA stimulation improved both chemoreflex responses in AngII rats (84±15 bpm and 32±5 mmHg, P&lt;0.05). In contrast, EA stimulation had no significant effect on control group. These results indicate that EA stimulation improves arterial baroreflex function and chemoreflex sensitivity in conditions of high Ang II.

Time-dependent changes in autonomic control of splanchnic vascular resistance and heart rate in ANG II-salt hypertension

Previous studies suggest that ANG II-induced hypertension in rats fed a high-salt (HS) diet (ANG II-salt hypertension) has a neurogenic component dependent on an enhanced sympathetic tone to the splanchnic veins and independent from changes in sympathetic nerve activity to the kidney or hind limb. The purpose of this study was to extend these findings and test whether altered autonomic control of splanchnic resistance arteries and the heart also contributes to the neurogenic component. Mean arterial pressure (MAP), heart rate (HR), superior mesenteric artery blood flow, and mesenteric vascular resistance (MVR) were measured during 4 control days, 14 days of ANG II delivered subcutaneously (150 ng·kg(-1)·min(-1)), and 4 days of recovery in conscious rats fed a HS (2% NaCl) or low-salt (LS; 0.1% NaCl) diet. Autonomic effects on MAP, HR, and MVR were assessed by acute ganglionic blockade with hexamethonium (20 mg/kg iv) on day 3 of control, days 1, 3, 5, 7, 10, and 13 of ANG II, and day 4 of recovery. MVR increased during ANG II infusion in HS and LS rats but remained elevated only in HS rats. Additionally, the MVR response to hexamethonium was enhanced on days 10 and 13 of ANG II selectively in HS rats. Compared with LS rats, HR in HS rats was higher during the 2nd wk of ANG II, and its response to hexamethonium was greater on days 7, 10, and 13 of ANG II. These results suggest that ANG II-salt hypertension is associated with delayed changes in autonomic control of splanchnic resistance arteries and the heart.

Involvement of cytochrome P-450 1B1 in renal dysfunction, injury, and inflammation associated with angiotensin II-induced hypertension in rats

We investigated the contribution of cytochrome P-450 1B1 (CYP1B1) to renal dysfunction and organ damage associated with ANG II-induced hypertension in rats. ANG II (300 ng·kg(-1)·min(-1)) or vehicle were infused for 2 wk, with daily injections of a selective CYP1B1 inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS; 300 μg/kg ip), or its vehicle. ANG II increased blood pressure and renal CYP1B1 activity that were prevented by TMS. ANG II also increased water intake and urine output, decreased glomerular filtration rate, increased urinary Na(+) and K(+) excretion, and caused proteinuria, all of which were prevented by TMS. ANG II infusion caused hypertrophy, endothelial dysfunction, and increased reactivity of renal and interlobar arteries to vasoconstrictor agents and renal vascular resistance and interstitial fibrosis as indicated by accumulation of α-smooth muscle actin, fibronectin, and collagen, and inflammation as indicated by increased infiltration of CD-3(+) cells; these effects were inhibited by TMS. ANG II infusion also increased production of reactive oxygen species (ROS) and activities of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src that were prevented by TMS. TMS alone had no effect on any of the above parameters. These data suggest that CYP1B1 contributes to the renal pathophysiological changes associated with ANG II-induced hypertension, most likely via increased ROS production and activation of ERK1/2, p38 MAPK, and c-Src and that CYP1B1 could serve as a novel target for treating renal disease associated with hypertension.

Inhibition of Cytochrome P450 1B1 Activity Prevents Renal Injury and Inflammation Associated with Angiotensin II‐Induced Hypertension in Rats

Previously, we reported that inhibition of cytochrome P450 (CYP) 1B1 activity with 2,4,3′,5′-tetramethoxystilbene (TMS) prevents the development of angiotensin II (Ang II)-induced hypertension and associated cardiovascular hypertrophy, decreased GFR and proteinuria in rats. This study was conducted to investigate the effect of TMS on renal injury and inflammation caused by Ang II-induced hypertension in rats. Infusion of Ang II (300 ng/kg/min) by miniosmotic pump increased systolic blood pressure (124 ± 3 mmHg to 206 ± 13 mmHg, P < 0.05), which was prevented by concurrent injections of TMS (300 μg/kg, i.p. daily). Ang II-induced increase in blood pressure was associated with renal interstitial fibrosis as demonstrated by increased immunoreactive staining of α-smooth muscle actin and fibronectin, as well as transforming growth factor-β, a signal transduction molecule involved in renal fibrosis. Infusion of Ang II also increased glomerular infiltration of T-cells, as indicated by CD-3-positively stained cells, as well as hypertrophy of intrarenal arteries. The renal injury and inflammation caused by Ang II was prevented by co-administration of TMS. These data suggest that the renal injury and inflammation observed with Ang II-induced hypertension is dependent on CYP1B1 activity. Moreover, CYP1B1 could serve as a novel target for the development of drugs to treat renal disease associated with hypertension.

Prorenin and Its Ancient Receptor

Prorenin, the precursor of renin, has for a long time been considered to be functionally inert, despite high circulating concentrations. Plasma levels of prorenin usually exceed renin levels and are even further increased under pathological conditions, such as in diabetes. First support of a functional significance of prorenin came from studies in transgenic rats, which overexpress a renin gene in the liver resulting in high circulating prorenin levels.1 Without systemic renin–angiotensin system activation and with normal blood pressure, these animals developed a vascular phenotype reminiscent of malignant hypertension. Moreover, in a series of elegant experiments, Ichihara et al2–4 supported the concept of prorenin as a relevant factor in the pathophysiology of diseases, such as hypertension and diabetes, by using a peptide that binds and inhibits the activation of the precursor. Thus, this peptide attenuated nephropathy in streptozotocin-induced diabetes2 and cardiac fibrosis in stroke-prone spontaneously hypertensive rats.3 By successfully repeating the streptozotocin experiment in mice deficient for the angiotensin II type 1a (AT1a) receptor, they reported recently that these effects are independent of renin activation or angiotensin II generation and concluded that the peptide, in fact, inhibits the interaction of prorenin with a receptor.4 In this issue of Hypertension , Saris et al5 contribute further evidence for an angiotensin-independent action of prorenin on cells by showing that neonatal cardiomyocytes respond to purified prorenin by p38 mitogen-activated protein (MAP) kinase phosphorylation and broad changes in gene expression partially also caused by the MAP kinase activation. Because the authors had shown before that these cells are not able to synthesize angiotensin II after the addition of prorenin, they consider the observed effects as being …

Angiotensin-converting enzyme inhibitors: a new mechanism of action.

Angiotensin-converting enzyme (ACE) inhibitors are valuable agents for the treatment of hypertension, heart failure, and other cardiovascular and renal diseases. The cardioprotective effects of ACE inhibitors are mediated by blockade of both conversion of angiotensin (Ang) I to Ang II and kinin hydrolysis. Here, we report a novel mechanism that may explain the cardiac antifibrotic effect of ACE inhibition, involving blockade of the hydrolysis of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). To study the role of Ac-SDKP in the therapeutic effects of the ACE inhibitor captopril, we used a model of Ang II-induced hypertension in rats treated with the ACE inhibitor either alone or combined with a blocking monoclonal antibody (mAb) to Ac-SDKP. These hypertensive rats had left ventricular hypertrophy (LVH) as well as increases in cardiac fibrosis, cell proliferation, transforming growth factor-beta (TGF-beta) expression, and phosphorylation of Smad2 (P-Smad2), a signaling mediator of the effects of TGF-beta. The ACE inhibitor did not decrease either blood pressure or LVH; however, it significantly decreased LV collagen from 13.3+/-0.9 to 9.6+/-0.6 microg/mg dry wt (P<0.006), and this effect was blocked by the mAb (12.1+/-0.6; P<0.034, ACE inhibitor versus ACE inhibitor+mAb). In addition, analysis of interstitial collagen volume fraction and perivascular collagen (picrosirius red staining) showed a very similar tendency. Likewise, the ACE inhibitor significantly decreased LV monocyte/macrophage infiltration, cell proliferation, and TGF-beta expression, and these effects were blocked by the mAb. Ang II increased Smad2 phosphorylation 3.2+/-0.9-fold; the ACE inhibitor lowered this to 0.6+/-0.1-fold (P<0.001), and the mAb blocked this decrease to 2.1+/-0.3 (P<0.001, ACE inhibitor versus ACE inhibitor+mAb). Similar findings were seen when the ACE inhibitor was replaced by Ac-SDKP. We concluded that in Ang II-induced hypertension, the cardiac antifibrotic effect of ACE inhibitors is a result of the inhibition of Ac-SDKP hydrolysis, resulting in a decrease in cardiac cell proliferation (probably fibroblasts), inflammatory cell infiltration, TGF-beta expression, Smad2 activation, and collagen deposition.

Endothelin mediates superoxide production in angiotensin II-induced hypertension in rats

Angiotensin II and endothelin-1 (ET) are two hormones involved in cardiovascular diseases and well known for their capacity to induce free radical generation in vascular and cardiac tissues. In addition to its prooxidative effect, angiotensin II can increase the synthesis of ET-1 in vascular smooth muscle cells (VSMC). Our objective was to determine whether the ET-1 synthesis in VSMC is involved in angiotensin II-induced superoxide anion production in rats. Our results show that treatments of isolated VSMC with angiotensin II and ET increased superoxide. However, this increase occurred in a bimodal fashion for angiotensin II with a fast transient production (10 min) and a late sustained production (6 h), while ET-1 induced superoxide formation after a delay of 6 h. LU302872 and BQ-123, a nonselective and a selective ETA receptor antagonists, respectively, prevented angiotensin II-induced superoxide anion production only during the late phase. In contrast, BQ-3020, a selective ETB receptor antagonist, had no effect. In vivo, LU302872 reduced the aortic superoxide production induced by angiotensin II administered for 12 days. In conclusion, our results suggest that the superoxide generation induced by chronic angiotensin II infusion may be mediated by ET-1 acting on ETA receptors in VSMC in vitro. Furthermore, this effect appears to contribute to the excess superoxide production during the chronic activation of the renin-angiotensin system in vivo.

Role of ET(A) receptors in experimental ANG II-induced hypertension in rats.

The objectives were to determine if ANG II-induced hypertension is maintained by activation of endothelin type A (ET(A)) receptors by endogenous ET-1 and if this effect is influenced by salt intake. Male rats were maintained on high sodium intake (HS; 6 meq/day) or on normal sodium intake (NS; 2 meq/day). Hypertension was produced by intravenous infusion of ANG II (5 ng/min) for 15 days. Five-day oral dosing with the selective ET(A)-receptor antagonist ABT-627 (~2 mg. kg(-1). day(-1)) reduced mean arterial pressure (MAP) to baseline levels in rats on HS receiving ANG II infusion, but it did not affect MAP in normotensive HS controls. In rats on NS, ABT-627 only transiently decreased MAP in rats receiving ANG II and slightly reduced MAP in normotensive controls. ABT-627 produced mild retention of sodium and water in NS rats receiving ANG II, but not in any other group. These results indicate that ET-1 plays a role in ANG II-induced hypertension via activation of ET(A) receptors and that this role is more prominent in rats on HS.