Amyloid beta accumulation in TgF344-AD rats is associated with reduced cerebral capillary endothelial Kir2.1 expression and neurovascular uncoupling.

Alzheimer's disease (AD) exerts a tremendous socio-economic burden worldwide. Although reduced cerebral blood flow is an early and persistent symptom that precedes the loss of cognitive function in AD, the underlying molecular and cellular mechanisms remain unclear. The present study investigated whether capillary endothelial inward rectifier potassium 2 (Kir2.1) expression is reduced in TgF344-AD (AD) rats and contributes to neurovascular uncoupling and cognitive deficits in AD. Three- to fourteen-month-old AD rats expressing mutant human APP and PS1 and age-matched wild-type (WT) F344 rats were studied. AD rats exhibited higher amyloid beta (Aß) expression in the brain as early as 3 months of age and amyloid plaques by 4 months of age. Functional hyperemic responses induced by whisker stimulation were impaired at 4 months of age, which were exacerbated in 6-month- and 14-month-old AD rats. The expression of Kir2.1 protein was significantly lower in the brains of 6-month-old AD versus WT rats, and Kir2.1 coverage was lower in the cerebral microvasculature of AD than in WT rats. Aß1-42 reduced the Kir2.1 expression in cultured capillary endothelial cells. Cerebral parenchymal arterioles with attached capillaries exhibited a reduced vasodilator in response to 10 mM K+ applied to capillaries, and constricted less following administration of a Kir2.1 channel blocker, compared to WT vessels. These results indicate that capillary endothelial Kir2.1 expression is reduced and contributes to impaired functional hyperemia in AD rats at early ages, perhaps secondary to elevated Aß expression.

VASCULAR AND RENAL MECHANISMS OF PREECLAMPSIA.

Preeclampsia (PE) is a multisystem obstetric disorder that affects 2-10% of pregnancies worldwide and it is a leading cause of maternal and fetal morbidity and mortality. The etiology of PE development is not clearly delineated, but since delivery of the fetus and placenta often leads to symptom resolution in the most cases of PE, it is hypothesized that the placenta is the inciting factor of the disease. Current management strategies for PE focus on treating the maternal symptoms to stabilize the mother in an attempt to prolong the pregnancy. However, the efficacy of this management strategy is limited. Therefore, identification of novel therapeutic targets and strategies is needed. Here, we provide a comprehensive overview of the current state of knowledge regarding mechanisms of vascular and renal pathophysiology during PE and discuss potential therapeutic targets directed at improving maternal vascular and renal function.

Platelet Inhibition Prevents NLRP3 Inflammasome Activation and Sepsis-Induced Kidney Injury.

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1ß and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8-10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1ß and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.

Interferon γ neutralization reduces blood pressure, uterine artery resistance index, and placental oxidative stress in placental ischemic rats.

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, and increased cytolytic natural killer cells (cNKs), which secrete interferon γ (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On gestation day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10 µg/kg of an anti-rat IFNγ monoclonal antibody. On GD 18, uterine artery resistance index (UARI) was measured via Doppler ultrasound and on GD 19, mean arterial pressure (MAP) was measured, animals were euthanized, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared with NP and was reduced in RUPP + anti-IFNγ. Placental ROS was also increased in RUPP rats compared with NP rats and was normalized in RUPP + anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared with NP rats and was reduced in RUPP + anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.

Acute kidney injury during pregnancy leads to increased sFlt-1 and sEng and decreased renal T regulatory cells in pregnant rats with HELLP syndrome.

BACKGROUND: The incidence of acute kidney injury (AKI) during pregnancy precedes a high maternal mortality rate of 20-40%. AKI during pregnancy has multiple etiologies; however, the more common are maternal hypertensive disorders, which include preeclampsia and HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome. Therefore, we sought to assess the impact of AKI on blood pressure, kidney injury, and anti-angiogenic factors during pregnancies with and without HELLP syndrome. METHODS: On gestational day (GD) 12, mini-osmotic pumps were inserted into a subset of normal pregnant (NP) rats infusing 4.7 µg/kg soluble fms-like tyrosine kinase-1 (sFlt-1) and 7 µg/kg soluble endoglin (sEng) to induce HELLP syndrome. On GD18, the renal pedicles were occluded for 45 min to induce AKI via bilateral ischemia reperfusion in a subset of NP (n = 18) or HELLP (n = 20) rats. Control NP (n = 20) and HELLP (n = 20) rats underwent a SHAM surgery on GD18. Plasma, urine, and maternal organs were saved for further analysis. Renal injury was assessed via renal histopathology, glomerular filtration rate (GFR), T cell infiltration, and assessment of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Data was measured via two-way analysis of variance with Tukey's test for post hoc analysis. RESULTS: Blood pressures were increased in HELLP+AKI rats (p = 0.0001); both NP+AKI and HELLP+AKI rats had increased lactate dehydrogenase (p < 0.0001) and aspartate aminotransferase levels (p < 0.0001), and decreased platelet levels (p < 0.001) vs. NP rats. HELLP+AKI (p = 0.002) and HELLP rats (p = 0.0002) had evidence of renal fibrosis vs. NP rats. GFR was decreased in HELLP+AKI (p = 0.01) rats vs. NP rats. Urinary KIM-1 was increased in NP+AKI rats vs. NP (p = 0.003) and HELLP rats (p = 0.01). HELLP+AKI rats had increased urinary KIM-1 vs. NP (p = 0.0008) and HELLP rats (p = 0.004) and increased NGAL vs. HELLP rats (p = 0.002). HELLP+AKI rats had increased sFlt-1 (p = 0.009) vs. NP rats. NP+AKI (p = 0.02) and HELLP+AKI (p = 0.007) rats had increased sEng vs. NP rats. CD3+CD4+ T cells were significantly increased in HELLP+AKI rats vs. NP (p = 0.0002) and NP+AKI (p = 0.05) rats. T regulatory cells were significantly decreased in HELLP+AKI (p = 0.03) and NP+AKI (p = 0.02) rats vs. NP rats; there were no changes between groups in T helper 17 cells (p = 0.34). CONCLUSION: The findings in this study suggest that AKI during pregnancy contributes to increased blood pressure and biochemical markers for HELLP syndrome, creates an anti-angiogenic imbalance, and exacerbates kidney injury as shown on histopathology, GFR, and kidney injury markers.

Interleukin-17 signaling mediates cytolytic natural killer cell activation in response to placental ischemia.

T-helper (TH)17s, IL-17, and cytolytic natural killer cells (cNKs) are increased in preeclampsia and contribute to the hypertension, inflammation, and fetal growth restriction that occurs in response to placental ischemia in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. As IL-17 stimulates NK cytotoxicity in vitro, we tested the hypothesis that IL-17 inhibition in RUPP rats would decrease cNK activation as a mechanism to improve maternal and fetal outcomes. On gestation day (GD) 14, rats undergoing RUPP received a miniosmotic pump infusing IL-17RC (100 pg/day), a soluble IL-17 receptor (RUPP + IL-17RC). On GD19, mean arterial pressure (MAP) was measured in normal pregnant (NP), RUPP, and RUPP + IL-17RC rats (n = 10-12/group), animals were euthanized, and blood and tissues were collected for analysis. MAP was 30% higher in RUPP compared with NP (P < 0.0001) and was 12% lower in RUPP + IL-17RC (P = 0.0007 vs. RUPP). Placental cytolytic NK cells were 132% higher in RUPP than in NP (P = 0.04 vs. NP) and were normalized in RUPP + IL-17RC (P = 0.03 vs. RUPP). Placental levels of TNF-α, a cNK-secreted cytokine, and macrophage inflammatory protein-3α (MIP-3α), a cNK chemokine, were higher in RUPP vs. NP and lower after IL-17 blockade. Placental VEGF was lower in RUPP vs. NP and was normalized in RUPP + IL-17RC. In vitro cytolytic activity of RUPP placental NKs was higher compared with NP and was blunted in RUPP + IL-17RC NKs. Finally, both fetal weight and placental weight were lower in RUPP compared with NP, and were improved in RUPP + IL-17RC. These data identify IL-17 as a mediator of cNK activation in response to placental ischemia during pregnancy.

Hypertension in an Animal Model of HELLP Syndrome is Associated With Activation of Endothelin 1.

Women with hypertensive forms of pregnancy such as hemolysis-elevated liver enzymes-low platelet syndrome have increased circulating endothelin 1; however, the relationship between hypertension and endothelin 1 has not been studied. Using an animal model, we sought to determine whether there was an increased activation/dysfunction of endothelin 1, the effect of endothelin 1 receptor-A blockade on hypertension and other manifestations of hemolysis, elevated liver enzymes, and low platelets syndrome. On gestational day 12, timed-pregnant rats were infused with soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEndoglin; 4.7 and 7 µg/kg) via mini-osmotic pumps for 8 days. A subset of rats were treated with receptor-A antagonist (ABT-627, 5mg/kg) for 8 days. Rats with hemolysis-elevated liver enzymes-low platelet syndrome had significantly increased hypertension (P = .0001), circulating endothelin 1 (P = .03), and a significant 3.3- and 7.2-fold increase in preproendothelin messenger RNA (mRNA) expression in the placenta and liver (P = .01 and .04). Urinary protein:creatinine ratio was significantly increased in these animals (P = .0007), and circulating factors from these rats stimulated a significant increase in endothelial cell secretion of endothelin 1 (P = .001) in an in vitro assay. Blockade of the endothelin 1 receptor A significantly decreased hypertension (P = .001), circulating endothelin 1, and interleukin 17 (P = .004 and .003), placental preproendothelin mRNA expression (P = .016), and urinary protein:creatinine ratio (P = .007) in rats with hemolysis-elevated liver enzymes-low platelet syndrome. Blockade of the endothelin 1 receptor A significantly decreased hemolysis (P = .009), liver enzymes (P = .011), and significantly increased platelet levels (P = .03) and decreased circulating CD4+ and CD8+ T lymphocytes (P = .0004 and .0001) in rats infused with sFlt-1 and sEndoglin. These data support the hypothesis that endothelin 1 activation has a critical role in pathophysiology of as hemolysis-elevated liver enzymes-low platelet syndrome.

Role of 20-HETE in the impaired myogenic and TGF responses of the Af-Art of Dahl salt-sensitive rats.

The present study examined whether 20-HETE production is reduced in the renal vasculature and whether this impairs myogenic or tubuloglomerular feedback (TGF) responses of the afferent arteriole (Af-Art). The production of 20-HETE was 73% lower in renal microvessels of Dahl salt-sensitive rats (SS) rats than in SS.5(BN) rats, in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes was transferred into the SS genetic background. The luminal diameter of the Af-Art decreased by 14.7 ± 1.5% in SS.5(BN) rats when the perfusion pressure was increased from 60 to 120 mmHg, but it remained unaltered in SS rats. Administration of an adenosine type 1 receptor agonist (CCPA, 1 µM) reduced the diameter of the Af-Art in the SS.5(BN) rats by 44 ± 2%, whereas the diameter of the Af-Art of SS rats was unaltered. Autoregulation of renal blood flow (RBF) and glomerular capillary pressure (PGC) was significantly impaired in SS rats but was intact in SS.5(BN) rats. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 µM), completely blocked the myogenic and adenosine responses in the Af-Art and autoregulation of RBF and PGC in SS.5(BN) rats, but it had no effect in SS rats. These data indicate that a deficiency in the formation of 20-HETE in renal microvessels impairs the reactivity of the Af-Art of SS rats and likely contributes to the development of hypertension induced renal injury.

Hypertension in an experimental model of systemic lupus erythematosus occurs independently of the renal nerves.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder with prevalent hypertension and renal injury. In this study, we tested whether the renal nerves contribute to the development of hypertension in an established mouse model of SLE (NZBWF1). Female SLE and control (NZW/LacJ) mice were subjected to either bilateral renal denervation or a sham procedure at 32 wk of age. Two weeks later, blood pressure was assessed in conscious mice using carotid artery catheters. Blood pressure was higher in SLE mice compared with controls, as previously reported; however, blood pressure was not altered in the denervated SLE or control mice. The development of albuminuria was markedly blunted in denervated SLE mice; however, glomerulosclerosis was increased. Renal denervation reduced renal cortical expression of monocyte-chemoattractant protein in SLE mice but did not significantly alter renal monocyte/macrophage infiltration. Renal cortical TNF-α expression was also increased in sham SLE mice, but this was not impacted by denervation. This study suggests that the renal nerves do not have a significant role in the pathogenesis of hypertension, but have a complex effect on the associated renal inflammation and renal injury.

Genetic basis of the impaired renal myogenic response in FHH rats.

This study examined the effect of substitution of a 2.4-megabase pair (Mbp) region of Brown Norway (BN) rat chromosome 1 (RNO1) between 258.8 and 261.2 Mbp onto the genetic background of fawn-hooded hypertensive (FHH) rats on autoregulation of renal blood flow (RBF), myogenic response of renal afferent arterioles (AF-art), K(+) channel activity in renal vascular smooth muscle cells (VSMCs), and development of proteinuria and renal injury. FHH rats exhibited poor autoregulation of RBF, while FHH.1BN congenic strains with the 2.4-Mbp BN region exhibited nearly perfect autoregulation of RBF. The diameter of AF-art from FHH rats increased in response to pressure but decreased in congenic strains containing the 2.4-Mbp BN region. Protein excretion and glomerular and interstitial damage were significantly higher in FHH rats than in congenic strains containing the 2.4-Mbp BN region. K(+) channel current was fivefold greater in VSMCs from renal arterioles of FHH rats than cells obtained from congenic strains containing the 2.4-Mbp region. Sequence analysis of the known and predicted genes in the 2.4-Mbp region of FHH rats revealed amino acid-altering variants in the exons of three genes: Add3, Rbm20, and Soc-2. Quantitative PCR studies indicated that Mxi1 and Rbm20 were differentially expressed in the renal vasculature of FHH and FHH.1BN congenic strain F. These data indicate that transfer of this 2.4-Mbp region from BN to FHH rats restores the myogenic response of AF-art and autoregulation of RBF, decreases K(+) current, and slows the progression of proteinuria and renal injury.

Temporal characterization of the development of renal injury in FHH rats and FHH.1BN congenic strains.

The present study examined the effect of transfer of portions of chromosome 1 that includes (FHH.1(BN) AR(+) strain) or excludes (control FHH.1(BN) AR(-) strain) a 4.3-Mb region from the Brown Norway (BN) rat that restores the autoregulation (AR) of renal blood flow (RBF) on the development of hypertension and renal injury in congenic strains of Fawn Hooded Hypertensive (FHH) rats. FHH and control AR(-) rats exhibited poor autoregulation of RBF, and glomerular capillary pressure (Pgc) rose by 19 ± 2 mmHg in FHH rats when renal perfusion pressure (RPP) was increased from 100 to 150 mmHg. In contrast, RBF was well autoregulated in the AR(+) strain, and Pgc only increased by 3 ± 1 mmHg when RPP was increased over this range. Baseline mean arterial pressure (MAP) at 12 wk of age was similar in all strains and averaged 122 mmHg. MAP increased significantly in FHH rats and was significantly higher by 12 mmHg in 21-wk-old FHH rats than in the FHH.1(BN) congenic strains. Protein excretion rose from 5 ± 1 to 397 ± 29 mg/day in 6- vs. 21-wk-old FHH rats. In contrast, protein excretion only increased to 139 ± 21 mg/day in the control AR(-) strain, and it did not increase significantly in the AR(+) strain. Glomerular permeability to albumin was similar in all strains at 6 wk of age. It increased significantly in 9-wk-old FHH and control AR(-) rats, but not in the AR(+) strain. The levels of matrix metalloproteinase (MMP)-2 and transforming growth factor (TGF)-ß2 protein were significantly higher in the renal cortex of 9-wk-old FHH rats compared with the levels seen in the AR(+) strain. These data indicate that transfer of a 4.3-Mb region of BN chromosome 1 into the FHH genetic background improves autoregulation of RBF, normalizes Pgc, and slows the progression of renal disease.

Transfer of the CYP4A region of chromosome 5 from Lewis to Dahl S rats attenuates renal injury.

This study examined the effect of transfer of overlapping regions of chromosome 5 that includes (4A(+)) or excludes (4A(-)) the cytochrome P-450 4A (CYP4A) genes from the Lewis rat on the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and the development of hypertension-induced renal disease in congenic strains of Dahl salt-sensitive (Dahl S) rats. The production of 20-HETE was higher in the outer medulla of 4A(+) than in Dahl S or 4A(-) rats. Mean arterial pressure (MAP) rose to 190 +/- 7 and 185 +/- 3 mmHg in Dahl S and 4A(-) rats fed a high-salt (HS) diet for 21 days but only to 150 +/- 5 mmHg in the 4A(+) strain. Protein excretion increased to 423 +/- 40 and 481 +/- 37 mg/day in Dahl S and 4A(-) rats vs. 125 +/- 15 mg/day in the 4A(+) strain. Baseline glomerular capillary pressure (Pgc) was lower in 4A(+) rats (38 +/- 1 mmHg) than in Dahl S rats (42 +/- 1 mmHg). Pgc increased to 50 +/- 1 mmHg in Dahl S rats fed a HS diet, whereas it remained unaltered in 4A(+) rats (39 +/- 1 mmHg). Baseline glomerular permeability to albumin (P(alb)) was lower in 4A(+) rats (0.19 +/- 0.05) than in Dahl S or 4A(-) rats (0.39 +/- 0.02). P(alb) rose to approximately 0.61 +/- 0.03 in 4A(-) and Dahl S rats fed a HS diet for 7 days, but it remained unaltered in the 4A(+) rats. The expression of transforming growth factor-beta2 was higher in glomeruli of Dahl S rats than in 4A(+) rats fed either a low-salt (LS) or HS diet. Chronic administration of a 20-HETE synthesis inhibitor (HET0016; 10 mg.kg(-1).day(-1) sc) reversed the fall in MAP and renoprotection seen in 4A(+) rats. These results indicate that the introgression of the CYP4A genes from Lewis rats into the Dahl S rats increases the renal formation of 20-HETE and attenuates the development of hypertension and renal disease.

Role of endogenous CYP450 metabolites of arachidonic acid in maintaining the glomerular protein permeability barrier.

This study examined the metabolism of arachidonic acid (AA) by cytochrome P-450 enzymes in isolated glomeruli and the effects of selective inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatetraenoic acids (EETs) on glomerular permeability to albumin (P(alb)). Glomeruli avidly produced 20-HETE, EETs, dihydroxyeicosatetraenoic acids (diHETEs), and HETEs when incubated with exogenous AA. N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 microM) selectively inhibited the formation of 20-HETE by 95% and increased P(alb) from 0.00 +/- 0.08 to 0.73 +/- 0.10 (n = 43 glomeruli, 4 rats). Addition of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (20-5,14-HEDE; 1 microM) opposed the effects of HET0016 (10 microM) to increase P(alb) (0.21 +/- 0.10, n = 36 glomeruli, 4 rats). Preincubation of glomeruli with exogenous AA to increase basal production of 20-HETE had a similar effect. We also examined the effect of an epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MSPPOH; 5 microM), on P(alb). MSPPOH (5 microM) significantly increased P(alb) but had no effect on the synthesis of EETs in glomeruli incubated with AA. However, MSPPOH (5 microM) selectively reduced epoxygenase activity by 50% in glomeruli incubated without added AA. Pretreatment with 8,9-EET (100 nM) attenuated the effects of MSPPOH (5 microM) on P(alb). These results indicate that glomeruli produce 20-HETE, EETs, diHETEs, and HETEs and that endogenously formed 20-HETE and EETs play an essential role in the maintenance of the glomerular permeability barrier to albumin.

Contribution of prostanoid tp receptors to the pressor and intrarenal haemodynamic response to endothelin.

Previous studies have shown that endothelin (ET)-1 stimulates thromboxane (Tx)A(2) production and so we hypothesized that inhibiting prostanoid TP receptors would prevent the pressor and intrarenal haemodynamic response to an acute infusion of ET-1. 2. Male Sprague-Dawley rats were anaesthetized with Inactin (Sigma Chemical, St Louis, MO, USA; 50 mg/kg) and catheters were inserted into the femoral artery and vein for recording mean arterial pressure (MAP) and infusion of ET-1 and receptor antagonists, respectively. A jugular vein catheter was used for the infusion of bovine serum albumin (6.2% in saline) during surgery (1.25% bodyweight). The pressor response to a 1 h infusion of ET-1 (6 pmol/kg per min) was determined in rats that had been pretreated with vehicle (0.9% NaCl) or the TP receptor antagonist SQ29548 (2 mg/kg per h). Laser Doppler single-optic fibres were implanted in the left kidney for the measurement of medullary blood flow (MBF) and cortical blood flow (CBF). 3. Prostanoid TP receptor blockade completely inhibited the acute pressor response to ET-1; the change in MAP was 14 2% versus -3 4% in vehicle and SQ29548 groups, respectively (P<0.05). Endothelin-1 reduced CBF (-15.2 3.3%), a response that was not significantly changed by SQ29548 (-6.2 7.6%). Similarly, the ET-1-mediated response in MBF was not altered by the TP receptor antagonist (7.7 4.9 vs 6.5 5.2%). 4. To determine the influence of the ET(B) receptor in modulating the response to ET-1 during TP receptor blockade, additional groups were pretreated with A-192621, an ET(B) receptor-selective antagonist (10 mg/kg, i.v.). A-192621 potentiated the increase in MAP produced by ET-1 (32 5%; P<0.05 vs ET-1 alone). SQ29548 significantly inhibited, but did not completely block, the increase in MAP produced by ET-1 during ET(B) antagonist treatment (18 4%; P<0.05). Endothelin-1-induced decreases in CBF were significantly enhanced in rats that were pretreated with A-192621, whereas ET-1 also significantly decreased MBF following A-192621 treatment. During ET(B) receptor blockade, TP receptor inhibition had no effect on the ET-1-mediated response of CBF and MBF. 5. These results suggest that TP receptor activation is not involved in the renal haemodynamic responses to ET-1. However, TP receptor activation contributes to the acute pressor response to ET-1, but does not account for the potentiated increase in MAP during ET(B) receptor blockade.

Peroxisome proliferator-activated receptor-alpha activation reduces salt-dependent hypertension during chronic endothelin B receptor blockade.

Endothelin B (ETB) receptor stimulation inhibits sodium transport in a similar fashion as 20-HETE. Clofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist, increases protein expression of cytochrome P450 4A (CYP4A), which is responsible for 20-HETE synthesis in the kidney. Experiments were designed to determine whether clofibrate reduces hypertension associated with chronic ETB receptor blockade. Male Sprague-Dawley rats received either normal-salt (0.8% NaCl) or high-salt (8% NaCl) diet for 10 days. Female rats were fed a high-salt (8% NaCl) diet for 10 days. During the last 7 days, rats of both sexes were divided into 3 treatment groups: (1) clofibrate in drinking water (80 mg per day), (2) ETB receptor antagonist A-192621 in food (10 mg/kg per day), or (3) clofibrate and A-192621. During ETB receptor blockade, clofibrate had no effect on mean arterial pressure (MAP) under normal salt conditions. In contrast, clofibrate significantly inhibited the increase in MAP produced by A-192621 in rats fed a high-salt diet (34+/-3 versus 19+/-4 mm Hg; P <0.05). Similar results were observed in female rats administered A-192621 and fed a high-salt diet. ETB receptor blockade significantly decreased CYP4A protein expression in the renal cortex of rats on high salt. Clofibrate significantly increased renal cortical and medullary CYP4A protein expression in A-192621-treated male rats on high salt. Therefore, chronic PPAR-alpha agonist treatment reduces salt-dependent hypertension produced by ETB receptor blockade in male and female Sprague-Dawley rats. This suggests a possible relationship between ETB receptor activation and the maintenance of CYP4A protein expression in the kidney of rats fed a high-salt diet.

Arterial pressure response to the antioxidant tempol and ETB receptor blockade in rats on a high-salt diet.

We hypothesized that increased superoxide contributes to mean arterial pressure (MAP) regulation in male Sprague-Dawley rats fed a high-salt diet and/or during endothelin (ET(B)) receptor blockade. Four groups on either a normal- or a high-salt diet were studied for 1 week: (1) control; (2) tempol, a superoxide dismutase mimetic, in their drinking water (1 mmol/L); (3) A-192621, an ET(B) antagonist, in their food (10 mg/kg daily); or (4) both tempol and A-192621. Without ET(B) blockade, tempol had no effect on MAP (telemetry) in rats on the normal-salt diet but significantly reduced MAP in rats on the high-salt diet (100+/-3 vs 112+/-2 mm Hg, P<0.05). On the normal-salt diet, A-192621 increased MAP with or without tempol. Under high-salt conditions, tempol attenuated the increase in MAP produced by A-192621, but only during the initial days of treatment. Plasma 8-isoprostanes were increased in all rats on the high-salt diet and were further increased after 3 days of A-192621 but not after 7 days; tempol inhibited the increase produced by A-192621 but had no influence on the increase produced by high salt. H2O2 excretion was significantly higher in rats on a high-salt diet for the 7-day drug treatment compared with those on a normal-salt diet. Tempol further increased H2O2 excretion in rats on a high-salt diet, an effect accelerated in A-192621-treated rats. These data suggest that blood pressure lowering by tempol in rats on a high-salt diet may be unrelated to reductions in superoxide and that renal H2O2 may account for the limited ability of tempol to attenuate hypertension produced by ET(B) receptor blockade.

Targeting sources of superoxide and increasing nitric oxide bioavailability in hypertension.

Overproduction of oxygen free radicals, which is mainly mediated by superoxide, occurs in human hypertension and a wide variety of animal models. There are several important enzymatic sources of superoxide production, including NADPH oxidase, xanthine oxidase and uncoupled nitric oxide synthase. Superoxide levels are also controlled through endogenous antioxidant systems and superoxide dismutase is the primary antioxidant in the vascular system. Strategies have therefore focused on combating hypertension and vascular disease through the inhibition of superoxide-generating enzymes, and scavenging superoxide. While results from animal studies are promising, no consensus has been reached on identifying a drug target for the reliable and effective treatment of oxidative stress in hypertension.