Activating autoantibodies to the angiotensin II type I receptor play an important role in mediating hypertension in response to adoptive transfer of CD4+ T lymphocytes from placental ischemic rats

Hypertension in rats with chronic placental ischemia (reduced uterine perfusion pressure, RUPP) is associated with elevated inflammatory cytokines, agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and CD4(+) T cells; all of which are elevated in preclamptic women. Additionally, we have shown that adoptive transfer of RUPP CD4(+) T cells increases blood pressure, inflammatory cytokines, and sFlt-1. The objective of this study was to determine the long-term effects of RUPP CD4(+) T cells on AT1-AA, renal and systemic hemodynamics in pregnant rats. To answer this question CD4(+) T splenocytes were magnetically isolated on day 19 of gestation from control RUPP and normal pregnant (NP) rats and injected into a new group of NP rats at day 13 of gestation. On day 19 of gestation mean arterial pressure (MAP) and renal function (glomerular filtration rates, GFR) were analyzed and serum collected for AT1-AA analysis. To determine a role for AT1-AA to mediate RUPP CD4(+) T cell-induced blood pressure increases, MAP was analyzed in a second group of rats treated with AT1 receptor blockade losartan (10 mg·kg(-1)·day(-1)) and in a third group of rats treated with rituximab, a B cell-depleting agent (250 mg/kg) we have shown previously to decrease AT1-AA production in RUPP rats. MAP increased from 101 ± 2 mmHg NP to 126 ± 2 mmHg in RUPP rats (P < 0.001) and to 123 ± 1 mmHg in NP rats injected with RUPP CD4(+) T cells (NP+RUPP CD4(+)T cells) (P < 0.001). Furthermore, GFR decreased from 2.2 ml/min (n = 7) in NP rats to 1.0 ml/min (n = 5) NP+RUPP CD4(+)T cell. Circulating AT1-AA increased from 0.22 ± 0.1 units in NP rats to 13 ± 0.7 (P < 0.001) units in NP+RUPP CD4(+)T cell-treated rats but decreased to 8.34 ± 1 beats/min in NP+RUPP CD4(+) T cells chronically treated with rituximab. Hypertension in NP+RUPP CD4(+)T cell group was attenuated by losartan (102 ± 4 mmHg) and with B cell depletion (101 ± 5 mmHg). Therefore, we conclude that one mechanism of hypertension in response to CD4(+) T lymphocytes activated during placental ischemia is via AT1 receptor activation, potentially via AT1-AA during pregnancy.     

Agonistic Autoantibodies to the Angiotensin II Type I Receptor Cause Pathophysiologic Characteristics of Preeclampsia

BackgroundPreeclampsia (PE), new-onset hypertension with proteinuria during pregnancy, is associated with increased reactive oxygen species, the vasoactive peptide endothelin-1 (ET-1), T and B lymphocytes, soluble antiangiogenic factors sFlt-1 and sEndoglin (sFlt-1 and sEng), and agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA).ObjectivesOne important area of investigation for our laboratory was to determine what role AT1-AA plays in the pathophysiology associated with PE.MethodsTo achieve this goal, we examined the effect of AT1-AA suppression on hypertension in response to placental ischemia as well as the effect of AT1-AA on increased blood pressure, ET-1, reactive oxygen species, and sFlt-1 in normal pregnant rats (NP).ResultsWe demonstrated reductions in uterine perfusion pressure (RUPP) to be a stimulus for AT1-AA during pregnancy. We utilized the technique of B-cell depletion to suppress circulating AT1-AA in RUPP rats and found that AT1-AA suppression in RUPP rats was associated with lower blood pressure and ET-1 activation. To determine a role for AT1-AA to mediate hypertension during pregnancy, we infused purified rat AT1-AA (1:50) into NP rats, and analyzed blood pressure and soluble factors. We consistently found that AT1-AA infused rats had significantly increased AT1-AA and blood pressure above NP rats. This hypertension was associated with significantly increased ET-1 in renal cortices (11-fold) and placenta (4-fold), and there was an approximately 2- to 3-fold increase in placental oxidative stress. Furthermore, antiangiogenic factors sFlt-1 and sEng were significantly increased in the AT1-AA induced hypertensive group compared with the NP controls.ConclusionsCollectively, these data indicated an important role for AT1-AA stimulated in response to placental ischemia that caused hypertension during pregnancy.     

Hypertension in response to CD4(+) T cells from reduced uterine perfusion pregnant rats is associated with activation of the endothelin-1 system

We have shown that adoptive transfer of CD4(+) T cells from placental ischemia (reduction in uteroplacental perfusion, RUPP) rats causes hypertension and elevated inflammatory cytokines during pregnancy. In this study we tested the hypothesis that adoptive transfer of RUPP CD4(+) T cells was associated with endothelin-1 activation as a mechanism to increase blood pressure during pregnancy. CD4(+) T cells from RUPP or normal pregnant (NP) rats were adoptively transferred into NP rats on gestational day 13. Mean arterial pressure (MAP) was analyzed on gestational day 19, and tissues were collected for endothelin-1 analysis. MAP increased in placental ischemic RUPP rats versus NP rats (124.1 ± 3 vs. 96.2 ± 3 mmHg; P = 0.0001) and increased in NP recipients of RUPP CD4(+) T cells (117.8 ± 2 mmHg; P = 0.001 compared with NP). Adoptive transfer of RUPP CD4(+) T cells increased placental preproendothelin-1 mRNA 2.1-fold compared with NP CD4(+) T cell rats and 1.7-fold compared with NP. Endothelin-1 secretion from endothelial cells exposed to NP rat serum was 52.2 ± 1.9 pg·mg(-1)·ml(-1), 77.5 ± 4.3 pg·mg(-1)·ml(-1) with RUPP rat serum (P = 0.0003); 47.2 ± .16 pg·mg(-1)·ml(-1) with NP+NP CD4(+) T cell serum, and 62.2 ± 2.1 pg·mg(-1)·ml(-1) with NP+RUPP CD4(+) T cell serum (P = 0.002). To test the role of endothelin-1 in RUPP CD4(+) T cell-induced hypertension, pregnant rats were treated with an endothelin A (ET(A)) receptor antagonist (ABT-627, 5 mg/kg) via drinking water. MAP was 92 ± 2 mmHg in NP+ET(A) blockade and 108 ± 3 mmHg in RUPP+ET(A) blockade; 95 ± 5 mmHg in NP+NP CD4(+) T cells+ET(A) blockade and 102 ± 2 mmHg in NP+RUPP CD4(+) T cells+ET(A) blockade. These data indicate the importance of endothelin-1 activation to cause hypertension via chronic exposure to activated CD4(+) T cells in response to placental ischemia.     

Nitric oxide synthase, ADMA, SDMA, and nitric oxide activity in the paraventricular nucleus throughout the etiology of renal wrap hypertension

Within the paraventricular nucleus (PVN), there is a balance between the excitatory and inhibitory neurotransmitters that regulate blood pressure; in hypertension, the balance shifts to enhanced excitation. Nitric oxide (NO) is an atypical neurotransmitter that elicits inhibitory effects on cardiovascular function. We hypothesized that reduced PVN NO led to elevations in blood pressure during both the onset and sustained phases of hypertension due to decreased NO synthase (NOS) and increased asymmetrical dimethylarginine (ADMA; an endogenous NOS inhibitor) and symmetric dimethylarginine (SDMA). Elevated blood pressure, in response to PVN bilateral microinjections of a NO inhibitor, nitro-L-arginine methyl ester, was blunted in renal wrapped rats during the onset of hypertension (day 7) and sustained renal wrap hypertension (day 28) compared with sham-operated rats. Adenoviruses (Ad) encoding endothelial NOS (eNOS) or LacZ microinjected into the PVN [1 × 10(9) plaque-forming units, bilateral (200 nl/site)] reduced mean arterial pressure compared with control (Day 7, Ad LacZ wrap: 144 ± 7 mmHg and Ad eNOS wrap: 117 ± 5 mmHg, P ≤ 0.05) throughout the study (Day 28, Ad LacZ wrap: 123 ± 1 mmHg and Ad eNOS wrap: 108 ± 4 mmHg, P ≤ 0.05). Western blot analyses of PVN NOS revealed significantly lower PVN neuronal NOS during the onset of hypertension but not in sustained hypertension. Reduced SDMA was found in the PVN during the onset of hypertension; however, no change in ADMA was observed. In conclusion, functional indexes of NO activity indicated an overall downregulation of NO in renal wrap hypertension, but the mechanism by which this occurs likely differs throughout the development of hypertension.     

Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production

Insulin stimulates production of NO in vascular endothelium via activation of phosphatidylinositol (PI) 3-kinase, Akt, and endothelial NO synthase. We hypothesized that insulin resistance may cause imbalance between endothelial vasodilators and vasoconstrictors (e.g., NO and ET-1), leading to hypertension. Twelve-week-old male spontaneously hypertensive rats (SHR) were hypertensive and insulin resistant compared with control Wistar-Kyoto (WKY) rats (systolic blood pressure 202 +/- 11 vs. 132 +/- 10 mmHg; fasting plasma insulin 5 +/- 1 vs. 0.9 +/- 0.1 ng/ml; P < 0.001). In WKY rats, insulin stimulated dose-dependent relaxation of mesenteric arteries precontracted with norepinephrine (NE) ex vivo. This depended on intact endothelium and was blocked by genistein, wortmannin, or N(omega)-nitro-l-arginine methyl ester (inhibitors of tyrosine kinase, PI3-kinase, and NO synthases, respectively). Vasodilation in response to insulin (but not ACh) was impaired by 20% in SHR (vs. WKY, P < 0.005). Preincubation of arteries with insulin significantly reduced the contractile effect of NE by 20% in WKY but not SHR rats. In SHR, the effect of insulin to reduce NE-mediated vasoconstriction became evident when insulin pretreatment was accompanied by ET-1 receptor blockade (BQ-123, BQ-788). Similar results were observed during treatment with the MEK inhibitor PD-98059. In addition, insulin-stimulated secretion of ET-1 from primary endothelial cells was significantly reduced by pretreatment of cells with PD-98059 (but not wortmannin). We conclude that insulin resistance in SHR is accompanied by endothelial dysfunction in mesenteric vessels with impaired PI3-kinase-dependent NO production and enhanced MAPK-dependent ET-1 secretion. These results may reflect pathophysiology in other vascular beds that directly contribute to elevated peripheral vascular resistance and hypertension.     

IL-17-mediated oxidative stress is an important stimulator of AT1-AA and hypertension during pregnancy

Preeclampsia is associated with autoimmune cells T(H)17, secreting interleukin-17, autoantibodies activating the angiotensin II type I receptor (AT1-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic IL-17 increases blood pressure by stimulating ROS and AT1-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = 20), NP+IL-17 (n = 12), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = 7) (a superoxide dismutase mimetic that scavenges ROS), and NP+IL-17+tempol (n = 11). IL-17 (150 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day 19 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT1-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to 123 ± 3 mmHg in IL-17-infused NP rats. Urinary isoprostane increased from 1,029 ± 1 in NP to 3,526 ± 2 pg·mg(-1)·day(-1) in IL-17-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-1)·min(-1) (n = 4) in NP and 702 ± 5 (n = 5) RLU·ml(-1)·min(-1) in IL-17-treated rats. Importantly, AT1-AA increased from 0.41 ± 0.05 beats/min in NP rats (n = 8) to 18.4 ± 1 beats/min in IL-17 rats (n = 12). Administration of tempol attenuated the hypertension (101 ± 3 mmHg) ROS (459 ± 5 RLU·ml(-1)·min(-1)) and blunted AT1-AAs (7.3 ± 0.6 beats/min) in NP+IL-17+tempol-treated rats. Additionally, AT1 receptor blockade inhibited IL-17-induced hypertension and placental oxidative stress. MAP was 105 ± 5 mmHg and ROS was 418 ± 5 RLU·ml(-1)·min(-1) in NP+IL 17-treated with losartan. These data indicate that IL-17 causes placental oxidative stress, which serves as stimulus modulating AT1-AAs that may play an important role in mediating IL-17-induced hypertension during pregnancy.     

Reactive oxygen species contribute to sleep apnea-induced hypertension in rats

In clinical studies, sleep apnea is associated with hypertension, oxidative stress, and increased circulating endothelin-1 (ET-1). We previously developed a model of sleep apnea by exposing rats to eucapnic intermittent hypoxia (IH-C) during sleep, which increases both blood pressure and plasma levels of ET-1. Because similar protocols in mice increase tissue and plasma markers of oxidative stress, we hypothesized that IH-C generation of reactive oxygen species (ROS) contributes to the development of ET-1-dependent hypertension in IH-C rats. To test this, male Sprague-Dawley rats were instrumented with indwelling blood pressure telemeters and drank either plain water or water containing the superoxide dismutase mimetic, Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1 mM). Mean arterial pressure (MAP) and heart rate (HR) were recorded for 3 control days and 14 treatment days with rats exposed 7 h/day to IH-C or air/air cycling (Sham). On day 14, MAP in IH-C rats treated with Tempol (107 +/- 2.29 mmHg) was significantly lower than in untreated IH-C rats (118 +/- 9 mmHg, P < 0.05). Tempol did not affect blood pressure in sham-operated rats (Tempol = 101 +/- 3, water = 101 +/- 2 mmHg). Immunoreactive ET-1 was greater in plasma from IH-C rats compared with plasma from sham-operated rats but was not different from Sham in Tempol-treated IH-C rats. Small mesenteric arteries from IH-C rats but not Tempol-treated IH-C rats had increased superoxide levels as measured by ferric cytochrome c reduction, lucigenin signaling, and dihydroethidium fluorescence. The data show that IH-C increases ET-1 production and vascular ROS levels and that scavenging superoxide prevents both. Thus oxidative stress appears to contribute to increases in ET-1 production and elevated arterial pressure in this rat model of sleep apnea-induced hypertension.     

Effect of selective ET(A) receptor blockade on natriuretic peptide gene expression in DOCA-salt hypertension

To determine the role of endothelin-1 (ET-1) in the upregulation of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) observed in deoxycorticosterone acetate (DOCA)-salt hypertension, the selective ET-1 type-A receptor (ET(A)) antagonist ABT-627 was chronically administered to normal controls and hypertensive rats. Chronic ET(A) blockade in DOCA-salt-treated rats prevented the increase in blood pressure and circulating natriuretic protein (NP) levels and partially prevented left ventricular hypertrophy. The changes observed in NP gene expression in the atria were not affected by ABT-627. In the ventricles, ABT-627 reduced NP gene expression. Rats receiving the ET(A) antagonist alone showed reduced left ventricular NP gene expression. ABT-627 did not affect ventricular collagen III gene expression but enhanced left ventricular alpha-myosin heavy chain expression. These findings suggest that in vivo, ventricular but not atrial NP production is regulated by ET-1. This difference in response between atrial and ventricular NP gene expression to ET(A) receptor blockade is similar to that observed by us after applying angiotensin-converting enzyme inhibitors in other hypertensive models. In general therefore, atrial NP gene expression may not be as sensitive to the endocrine environment as is ventricular NP gene expression.     

Sympathetic activation and nitric oxide function in early hypertension

The purpose of this study was to determine if tonic restrain of blood pressure by nitric oxide (NO) is impaired early in the development of hypertension. Impaired NO function is thought to contribute to hypertension, but it is not clear if this is explained by direct effects of NO on vascular tone or indirect modulation of sympathetic activity. We determined the blood pressure effect of NO synthase inhibition with N(ω)-monomethyl-l-arginine (L-NMMA) during autonomic blockade with trimethaphan to eliminate baroreflex buffering and NO modulation of autonomic tone. In this setting, impaired NO modulation of vascular tone would be reflected as a blunted pressor response to L-NMMA. We enrolled a total of 66 subjects (39 ± 1.3 yr old, 30 females), 20 normotensives, 20 prehypertensives (blood pressure between 120/80 and 140/90 mmHg), 17 hypertensives, and 9 smokers (included as "positive" controls of impaired NO function). Trimethaphan normalized blood pressure in hypertensives, suggesting increased sympathetic tone contributing to hypertension. In contrast, L-NMMA produced similar increases in systolic blood pressure in normal, prehypertensive, and hypertensive subjects (31 ± 2, 32 ± 2, and 30 ± 3 mmHg, respectively), whereas the response of smokers was blunted (16 ± 5 mmHg, P = 0.012). Our results suggest that sympathetic activity plays a role in hypertension. NO tonically restrains blood pressure by ～30 mmHg, but we found no evidence of impaired modulation by NO of vascular tone contributing to the early development of hypertension. If NO deficiency contributes to hypertension, it is likely to be through its modulation of the autonomic nervous system, which was excluded in this study.     

Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance, and markers of renal oxidative stress in the pregnant rat

Increased uterine artery resistance and angiogenic imbalance characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased free vascular endothelial growth factor (VEGF) are often associated with placental insufficiency and preeclampsia but not synonymous with hypertension. We hypothesized chronic reductions in utero-placental perfusion (RUPP) for 5 days (d) during either mid- (d12-d17) or late (d14-d19) gestation would have disparate effects on plasma sFlt-1 and VEGF levels and blood pressure. Five days of chronic RUPP was achieved by placement of silver clips on the abdominal aorta and ovarian arteries on either gestational d12 or d14. Arterial pressure was increased (P < 0.05) in RUPP vs. normal pregnant (NP) in both d17 (10%) and d19 (25%) groups, respectively. Circulating free VEGF was decreased (P < 0.05) and sFlt-1:VEGF ratio increased (P < 0.05) after 5 days of RUPP ending on d19 but not d17 compared with NP controls. Angiogenic imbalance, measured by an endothelial tube formation assay, was present in the d19 RUPP but not the d17 RUPP compared with age-matched NP rats. Five days of RUPP from days 14 to 19 decreased fetal and placental weights 10% (P < 0.01) compared with d19 NP controls. After 5 days of RUPP, from days 12 to 17 of pregnancy, fetal weights were 21% lighter (P < 0.01) compared with d17 NP controls, but placental weight was unchanged. These findings suggest that the timing during which placental insufficiency occurs may play an important role in determining the extent of alterations in angiogenic balance, fetal growth restriction, and the severity of placental ischemia-induced hypertension.     

Endogenous endothelin attenuates the pressor response to acute environmental stress via the ETA receptor

Clinical studies have documented an abrupt rise in plasma endothelin-1 (ET-1) coincident with an increase in mean arterial pressure (MAP) during the response to acute stress. We therefore examined the ET(A) and ET(B) receptor-dependent effects of ET-1 on the pressor response to acute environmental stress in ET-1-dependent hypertension. Stress was induced by administration of air jet pulses (3 min) in ET(B) receptor-deficient (ET(B) sl/sl) rats fed normal salt (NS; 0.8% NaCl), high salt (HS; 8% NaCl), and HS plus the ET(A) receptor antagonist ABT-627 (5 mg.kg(-1).day(-1)) on successive weeks. MAP was chronically monitored by telemetry. Total pressor response (area under the curve) was significantly reduced in ET(B) sl/sl rats maintained on a HS vs. NS diet [-6.8 mmHg (SD 18.7) vs. 29.3 mmHg (SD 8.1) x 3 min, P < 0.05]. Conversely, the total pressor response was augmented in both wild-type [34.2 mmHg (SD 29.2) x 3 min, P < 0.05 vs. NS] and ET(B) sl/sl rats [49.1 mmHg (SD 11.8) x 3 min, P < 0.05 vs. NS] by ABT-627. Blockade of ET(B) receptors in Sprague-Dawley rats caused an increase in basal MAP that was enhanced by HS and lowered by mixed ET(A)/ET(B) receptor antagonism; none of these treatments, however, had any effect on the pressor response. These data demonstrate that increasing endogenous ET-1 suppresses the pressor response to acute stress through ET(A) receptor activation in a genetic model of ET-1-dependent hypertension. These results are consistent with reports that ET-1 can attenuate sympathetically mediated responses.     

Hemodynamic responses to aortic depressor nerve stimulation in conscious L-NAME-induced hypertensive rats

The present study investigated whether baroreflex control of autonomic function is impaired when there is a deficiency in NO production and the role of adrenergic and cholinergic mechanisms in mediating reflex responses. Electrical stimulation of the aortic depressor nerve in conscious normotensive and nitro-l-arginine methyl ester (L-NAME)-induced hypertensive rats was applied before and after administration of methylatropine, atenolol, and prazosin alone or in combination. The hypotensive response to progressive electrical stimulation (5 to 90 Hz) was greater in hypertensive (-27 ± 2 to -64 ± 3 mmHg) than in normotensive rats (-17 ± 1 to -46 ± 2 mmHg), whereas the bradycardic response was similar in both groups (-34 ± 5 to -92 ± 9 and -21 ± 2 to -79 ± 7 beats/min, respectively). Methylatropine and atenolol showed no effect in the hypotensive response in either group. Methylatropine blunted the bradycardic response in both groups, whereas atenolol attenuated only in hypertensive rats. Prazosin blunted the hypotensive response in both normotensive (43%) and hypertensive rats (53%) but did not affect the bradycardic response in either group. Prazosin plus angiotensin II, used to restore basal arterial pressure, provided hemodynamic responses similar to those of prazosin alone. The triple pharmacological blockade abolished the bradycardic response in both groups but displayed similar residual hypotensive response in hypertensive (-13 ± 2 to -27 ± 2 mmHg) and normotensive rats (-10 ± 1 to -25 ± 3 mmHg). In conclusion, electrical stimulation produced a well-preserved baroreflex-mediated decrease in arterial pressure and heart rate in conscious l-NAME-induced hypertensive rats. Moreover, withdrawal of the sympathetic drive played a role in the reflex bradycardia only in hypertensive rats. The residual fall in pressure after the triple pharmacological blockade suggests the involvement of a vasodilatory mechanism unrelated to NO or deactivation of α(1)-adrenergic receptor.     

TNF-alpha inhibition reduces renal injury in DOCA-salt hypertensive rats

Studies suggest that the inflammatory cytokine TNF-alpha plays a role in the prognosis of end-stage renal diseases. We previously showed that TNF-alpha inhibition slowed the progression of hypertension and renal damage in angiotensin II salt-sensitive hypertension. Thus, we hypothesize that TNF-alpha contributes to renal inflammation in a model of mineralocorticoid-induced hypertension. Four groups of rats (n = 5 or 6) were studied for 3 wk with the following treatments: 1) placebo, 2) placebo + TNF-alpha inhibitor etanercept (1.25 mg.kg(-1).day(-1) sc), 3) deoxycorticosterone acetate + 0.9% NaCl to drink (DOCA-salt), or 4) DOCA-salt + etanercept. Mean arterial blood pressure (MAP) measured by telemetry increased in DOCA-salt rats compared with baseline (177 +/- 4 vs. 107 +/- 3 mmHg; P < 0.05), and TNF-alpha inhibition had no effect in the elevation of MAP in these rats (177 +/- 8 mmHg). Urinary protein excretion significantly increased in DOCA-salt rats compared with placebo (703 +/- 76 vs. 198 +/- 5 mg/day); etanercept lowered the proteinuria (514 +/- 64 mg/day; P < 0.05 vs. DOCA-salt alone). Urinary albumin excretion followed a similar pattern in each group. Urinary monocyte chemoattractant protein (MCP)-1 and endothelin (ET)-1 excretion were also increased in DOCA-salt rats compared with placebo (MCP-1: 939 +/- 104 vs. 43 +/- 7 ng/day, ET-1: 3.30 +/- 0.29 vs. 1.07 +/- 0.03 fmol/day; both P < 0.05); TNF-alpha inhibition significantly decreased both MCP-1 and ET-1 excretion (409 +/- 138 ng/day and 2.42 +/- 0.22 fmol/day, respectively; both P < 0.05 vs. DOCA-salt alone). Renal cortical NF-kappaB activity also increased in DOCA-salt hypertensive rats, and etanercept treatment significantly reduced this effect. These data support the hypothesis that TNF-alpha contributes to the increase in renal inflammation in DOCA-salt rats.     

Insulin infusion induces endothelin-1-dependent hypertension in rats

We previously showed that chronic insulin infusion induces insulin resistance, hyperendothelinemia, and hypertension in rats (C. C. Juan, V. S. Fang, C. F. Kwok, J. C. Perng, Y. C. Chou, and L. T. Ho. Metabolism 48: 465-471, 1999). Endothelin-1 (ET-1), a potent vasoconstrictor, is suggested to play an important role in maintaining vascular tone and regulating blood pressure, and insulin increases ET-1 production in vivo and in vitro. In the present study, BQ-610, a selective endothelin A receptor antagonist, was used to examine the role of ET-1 in insulin-induced hypertension in rats. BQ-610 (0.7 mg/ml; 0.5 ml/kg body wt) or normal saline was given intraperitoneally two times daily for 25 days to groups of rats infused with either saline or insulin (2 U/day via sc-implanted osmotic pumps), and changes in plasma levels of insulin, glucose, and ET-1 and the systolic blood pressure were measured over the experimental period, whereas changes in insulin sensitivity were examined at the end of the experimental period. Plasma insulin and ET-1 levels were measured by RIA, plasma glucose levels using a glucose analyzer, systolic blood pressure by the tail-cuff method, and insulin sensitivity by an oral glucose tolerance test. Our studies showed that insulin infusion caused sustained hyperinsulinemia in both saline- and BQ-610-injected rats over the infusion period. After pump implantation (2 wk), the systolic blood pressure was significantly higher in insulin-infused rats than in saline-infused rats in the saline-injected group (133 +/- 3.1 vs. 113 +/- 1.1 mmHg, P < 0.05) but not in the BQ-610-injected group (117 +/- 1.2 vs. 117 +/- 1.8 mmHg). Plasma ET-1 levels in both sets of insulin-infused rats were higher than in saline-infused controls (2.5 +/- 0.6 and 2.5 +/- 0.8 vs. 1.8 +/- 0.4 and 1.7 +/- 0.3 pmol/l, P < 0.05). Oral glucose tolerance tests showed that BQ-610 treatment did not prevent the insulin resistance caused by chronic insulin infusion. No significant changes were found in insulin sensitivity and blood pressure in saline-infused rats treated with BQ-610. In a separate experiment, insulin infusion induced the increase in arterial ET-1 content, hypertension, and subsequent plasma ET-1 elevation in rats. These results suggest that, in the insulin infusion rat model, ET-1 plays a mediating role in the development of hypertension, but not of insulin resistance.     

A perinatal nitric oxide donor increases renal vascular resistance and ameliorates hypertension and glomerular injury in adult fawn-hooded hypertensive rats

Enhancing perinatal nitric oxide (NO) availability persistently reduces blood pressure in spontaneously hypertensive rats. We hypothesize that this approach can be generalized to other models of genetic hypertension, for instance those associated with renal injury. Perinatal exposure to the NO donor molsidomine was studied in fawn-hooded hypertensive (FHH) rats, a model of mild hypertension, impaired preglomerular resistance, and progressive renal injury. Perinatal molsidomine increased urinary NO metabolite excretion at 8 wk of age, i.e., 4 wk after treatment was stopped (P < 0.05). Systolic blood pressure was persistently reduced after molsidomine (42-wk females: 118 +/- 3 vs. 141 +/- 5 and 36-wk males: 139 +/- 4 vs. 158 +/- 4 mmHg; both P < 0.001). Perinatal treatment decreased glomerular filtration rate (P < 0.05) and renal blood flow (P < 0.01) and increased renal vascular resistance (P < 0.05), without affecting filtration fraction, suggesting persistently increased preglomerular resistance. At 4 wk of age natriuresis was transiently increased by molsidomine (P < 0.05). Molsidomine decreased glomerulosclerosis (P < 0.05). Renal blood flow correlated positively with glomerulosclerosis in control (P < 0.001) but not in perinatally treated FHH rats. NO dependency of renal vascular resistance was increased by perinatal molsidomine. Perinatal enhancement of NO availability can ameliorate development of hypertension and renal injury in FHH rats. Paradoxically, glomerular protection by perinatal exposure to the NO donor molsidomine may be due to persistently increased preglomerular resistance. The mechanisms by which increased perinatal NO availability can persistently reprogram kidney function and ameliorate hypertension deserve further study.     

Systemic hemodynamic and regional blood flow changes in response to chronic reductions in uterine perfusion pressure in pregnant rats

Preeclampsia (PE) is associated with increased total peripheral resistance (TPR), reduced cardiac output (CO), and diminished uterine and placental blood flow. We have developed an animal model that employs chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats to generate a "preeclamptic-like" state during late gestation that is characterized by hypertension, proteinuria, and endothelial dysfunction. Although this animal model has many characteristics of human PE, the systemic hemodynamic and regional changes in blood flow that occur in response to chronic RUPP remains unknown. Therefore, we hypothesized that RUPP would decrease uteroplacental blood flow and CO, and increase TPR. Mean arterial pressure (MAP), CO, cardiac index (CI), TPR, and regional blood flow to various tissues were measured using radiolabeled microspheres in the following two groups of conscious rats: normal pregnant rats (NP; n = 8) and RUPP rats (n = 8). MAP was increased (132 +/- 4 vs. 99 +/- 3 mmHg) in the RUPP rats compared with the NP dams. The hypertension in RUPP rats was associated with increased TPR (2.15 +/- 0.02 vs. 0.98 +/- 0.08 mmHg x ml(-1) x min(-1)) and decreased CI (246 +/- 20 vs. 348 +/- 19 ml x min(-1) x kg(-1), P < 0.002) when contrasted with NP dams. Furthermore, uterine (0.16 +/- 0.03 vs. 0.38 +/- 0.09 ml x min(-1) x g tissue(-1)) and placental blood flow (0.30 +/- 0.08 vs. 0.70 +/- 0.10 ml x min(-1) x g tissue(-1)) were decreased in RUPP compared with the NP dams. These data demonstrate that the RUPP model of pregnancy-induced hypertension has systemic hemodynamic and regional blood flow alterations that are strikingly similar to those observed in women with PE.     

Nitric oxide in cyclosporine A-induced hypertension: endothelin receptors gene expression.

Cyclosporine A (CsA) is an immunosuppressive agent, which also causes hypertension. The effect of CsA on vascular responses was determined in spontaneously hypertensive rats and isolated rat aortic rings. Male rats weighing 250-300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by i.p. injection for 7 days. CsA administration produced a 27% increase (P < 0.001) in mean arterial pressure (MAP) which reached a plateau after 3 days. Conversely, the level of nitrate/nitrite, metabolites of nitric oxide (NO), decreased by 44% (P < 0.001) in the urine. In the presence of endothelin (ET) 10(-9) M, thoracic aortic rings from rats treated with olive oil, L-Arginine (L-Arg) or L-Arg+CsA showed a 100% increase (P < 0.001) in tension compared to the aortic rings from rats treated with CsA alone; aortic rings from rats treated with CsA alone did not respond to ET. The effects of CsA were reversed in both in vivo and in vitro by pretreatment with L-Arg (10 mg/kg/day ip), the precursor of NO. There were no changes in MAP and tension in rats treated with L-Arg alone. Possible explanation for lack of response to ET of aortic rings from CsA treated rats may be that CsA affected ET signalling pathway; ET receptors mRNA (messenger ribonucleic acid) gene expression was inhibited in aortic rings of rats treated with CsA. In summary, CsA inhibits endothelial NO formation, with resulting increases in MAP, and this inhibition can be overcome by parenteral administration of L-Arg.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)alpha-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)alpha and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARalpha ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARalpha activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARalpha ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARalpha knockout (KO) mice compared with its wild type (WT) litter mates (130+/-10 mmHg versus 107+/-4 mmHg). L-NAME (100mg/L p.o.), the inhibitor of NO production abolished the difference between PPARalpha KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8+/-1.4 pM/mg versus 8.3+/-0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46+/-6%, p<0.05) and a approximately 3 fold greater Ca2+-dependent NOS activity in kidney homogenates of untreated PPARalpha WT compared with the KO mice. Clofibrate, a PPARalpha ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19+/-4%, p<0.05), increased urinary NO excretion (4.06+/-0.53-7.07+/-1.59 microM/24 h; p<0.05) and reduced plasma 8-isoprostane level (45.8+/-15 microM versus 31.4+/-8 microM), and NADP(H) oxidase activity (16+/-5%). Implantation of DOCA pellet (20mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193+/-13 mmHg versus 130+/-12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARalpha activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

Renal medullary endothelin-1 is decreased in Dahl salt-sensitive rats

Although it is well established that the renal endothelin (ET-1) system plays an important role in regulating sodium excretion and blood pressure through activation of renal medullary ET(B) receptors, the role of this system in Dahl salt-sensitive (DS) hypertension is unclear. The purpose of this study was to determine whether the DS rat has abnormalities in the renal medullary endothelin system when maintained on a high sodium intake. The data indicate that Dahl salt-resistant rats (DR) on a high-salt diet had a six-fold higher urinary endothelin excretion than in the DR rats with low Na(+) intake (17.8 ± 4 pg/day vs. 112 ± 44 pg/day). In sharp contrast, urinary endothelin levels increased only twofold in DS rats in response to a high Na(+) intake (13 ± 2 pg/day vs. 29.8 ± 5.5 pg/day). Medullary endothelin concentration in DS rats on a high-Na(+) diet was also significantly lower than DR rats on a high-Na(+) diet (31 ± 2.8 pg/mg vs. 70.9 ± 5 pg/mg). Furthermore, DS rats had a significant reduction in medullary ET(B) receptor expression compared with DR rats while on a high-Na(+) diet. Finally, chronic infusion of ET-1 directly into the renal medulla blunted Dahl salt-sensitive hypertension. These data indicate that a decrease in medullary production of ET-1 in the DS rat could play an important role in the development of salt-sensitive hypertension observed in the DS rat.     

Imatinib ameliorates renal morphological changes in Cyp1a1-Ren2 transgenic rats with inducible ANG II-dependent malignant hypertension

The present study was performed to assess the effects of the platelet-derived growth factor (PDGF) receptor kinase inhibitor imatinib mesylate on the renal morphological changes occurring during the development of malignant hypertension in transgenic rats with inducible expression of the Ren2 gene [TGR(Cyp1a1Ren2)]. Arterial blood pressure was measured by radiotelemetry in male Cyp1a1-Ren2 rats during control conditions and during dietary administration of indole-3-carbinol (I3C; 0.3%) for 14 days to induce malignant hypertension. Rats induced with I3C (n = 5) had higher mean arterial pressures (178 ± 4 vs. 109 ± 2 mmHg, P < 0.001) and increased urinary albumin excretion (Ualb; 13 ± 5 vs. 0.6 ± 0.2 mg/day) compared with noninduced rats (n = 5). Chronic administration of imatinib (60 mg·kg(-1)·day(-1) in drinking water, n = 5) did not alter the magnitude of the hypertension (176 ± 8 mmHg) but prevented the increase in Ualb (1.6 ± 0.3 mg/day). Quantitative analysis of proliferating cell nuclear antigen using immunohistochemistry demonstrated increased proliferating cell number in cortical tubules (38 ± 5 vs. 18 ± 1 cells/mm(2)) and cortical interstitium (40 ± 7 vs. 13 ± 6 cells/mm(2)) of hypertensive rat kidneys. Renal cortical fibrosis evaluated by picrosirius red staining showed increased collagen deposition in kidneys of the hypertensive rats (1.6 ± 0.1 vs. 0.4 ± 0.1% of cortical area). Imatinib attenuated the increase in proliferating cell number in cortical tubules and interstitium (22 ± 5 vs. 38 ± 5 and 22 ± 6 vs. 40 ± 7 cells/mm(2), respectively) and reduced the degree of collagen deposition (0.8 ± 0.2 vs. 1.6 ± 0.1%) in the kidneys of hypertensive rats. These findings demonstrate that the renal pathological changes that occur during the development of malignant hypertension in Cyp1a1-Ren2 rats involve activation of PDGF receptor kinase.