Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1

It is well known that nonselective, nonsteroidal anti-inflammatory drugs inhibit renal renin production. Our previous studies indicated that angiotensin-converting enzyme inhibitor (ACEI)-mediated renin increases were absent in rats treated with a cyclooxygenase (COX)-2-selective inhibitor and in COX-2 -/- mice. The current study examined further whether COX-1 is also involved in mediating ACEI-induced renin production. Because renin increases are mediated by cAMP, we also examined whether increased renin is mediated by the prostaglandin E(2) receptor EP(2) subtype, which is coupled to G(s) and increases cAMP. Therefore, we investigated if genetic deletion of COX-1 or EP(2) prevents increased ACEI-induced renin expression. Age- and gender-matched wild-type (+/+) and homozygous null mice (-/-) were administered captopril for 7 days, and plasma and renal renin levels and renal renin mRNA expression were measured. There were no significant differences in the basal level of renal renin activity from plasma or renal tissue in COX-1 +/+ and -/- mice. Captopril administration increased renin equally [plasma renin activity (PRA): +/+ 9.3 +/- 2.2 vs. 50.1 +/- 10.9; -/- 13.7 +/- 1.5 vs. 43.9 +/- 6.6 ng ANG I x ml(-1) x h(-1); renal renin concentration: +/+ 11.8 +/- 1.7 vs. 35.3 +/- 3.9; -/- 13.0 +/- 3.0 vs. 27.8 +/- 2.7 ng ANG I x mg protein(-1) x h(-1); n = 6; P < 0.05 with or without captopril]. ACEI also increased renin mRNA expression (+/+ 2.4 +/- 0.2; -/- 2.1 +/- 0.2 fold control; n = 6-10; P < 0.05). Captopril led to similar increases in EP(2) -/- compared with +/+. The COX-2 inhibitor SC-58236 blocked ACEI-induced elevation in renal renin concentration in EP(2) null mice (+/+ 24.7 +/- 1.7 vs. 9.8 +/- 0.4; -/- 21.1 +/- 3.2 vs. 9.3 +/- 0.4 ng ANG I x mg protein(-1) x h(-1); n = 5) as well as in COX-1 -/- mice (SC-58236-treated PRA: +/+ 7.3 +/- 0.6; -/- 8.0 +/- 0.9 ng ANG I x ml(-1) x h(-1); renal renin: +/+ 9.1 +/- 0.9; -/- 9.6 +/- 0.5 ng ANG I x mg protein(-1) x h(-1); n = 6-7; P < 0.05 compared with no treatment). Immunohistochemical analysis of renin expression confirmed the above results. This study provides definitive evidence that metabolites of COX-2 rather than COX-1 mediate ACEI-induced renin increases. The persistent response in EP(2) nulls suggests involvement of prostaglandin E(2) receptor subtype 4 and/or prostacyclin receptor (IP).     

Adenosine A₁-receptor deficiency diminishes afferent arteriolar and blood pressure responses during nitric oxide inhibition and angiotensin II treatment

Adenosine mediates tubuloglomerular feedback responses via activation of A(1)-receptors on the renal afferent arteriole. Increased preglomerular reactivity, due to reduced nitric oxide (NO) production or increased levels of ANG II and reactive oxygen species (ROS), has been linked to hypertension. Using A(1)-receptor knockout (A(1)(-/-)) and wild-type (A(1)(+/+)) mice we investigated the hypothesis that A(1)-receptors modulate arteriolar and blood pressure responses during NO synthase (NOS) inhibition or ANG II treatment. Blood pressure and renal afferent arteriolar responses were measured in nontreated mice and in mice with prolonged N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or ANG II treatment. The hypertensive responses to L-NAME and ANG II were clearly attenuated in A(1)(-/-) mice. Arteriolar contractions to L-NAME (10(-4) mol/l; 15 min) and cumulative ANG II application (10(-12) to 10(-6) mol/l) were lower in A(1)(-/-) mice. Simultaneous treatment with tempol (10(-4) mol/l; 15 min) attenuated arteriolar responses in A(1)(+/+) but not in A(1)(-/-) mice, suggesting differences in ROS formation. Chronic treatment with L-NAME or ANG II did not alter arteriolar responses in A(1)(-/-) mice, but enhanced maximal contractions in A(1)(+/+) mice. In addition, chronic treatments were associated with higher plasma levels of dimethylarginines (asymmetrical and symmetrical) and oxidative stress marker malondialdehyde in A(1)(+/+) mice, and gene expression analysis showed reduced upregulation of NOS-isoforms and greater upregulation of NADPH oxidases. In conclusion, adenosine A(1)-receptors enhance preglomerular responses during NO inhibition and ANG II treatment. Interruption of A(1)-receptor signaling blunts l-NAME and ANG II-induced hypertension and oxidative stress and is linked to reduced responsiveness of afferent arterioles.     

Attenuated renal vascular responses to acute angiotensin II infusion in smooth muscle-specific Na+/Ca2+ exchanger knockout mice

Recent studies in smooth muscle-specific Na(+)/Ca(2+) exchanger-1 knockout (NCX1(sm-/-)) mice reveal reduced arterial pressure and impaired myogenic responses compared with heterozygous littermates. In this study, we determined renal function in male anesthetized NCX1(sm-/-) mice and NCX1 heterozygous (NCX1(+/-)) littermates before and during acute ANG II infusions. Systolic blood pressure in awake mice was lower in NCX1(sm-/-) mice compared with NCX1(+/-) mice (119 ± 4 vs. 131 ± 3 mmHg, P < 0.05). Acute ANG II infusions (5 ng·min(-1)·g(-1) body wt) increased mean arterial pressure in anesthetized NCX1(+/-) (109 ± 2 to 134 ± 3 mmHg, P < 0.001, n = 8) and NCX1(sm-/-) (101 ± 8 to 129 ± 8 mmHg, P < 0.01, n = 6) mice to a similar extent (Δ25 ± 1 vs. Δ28 ± 4 mmHg, P > 0.05). In response to ANG II infusions, PAH clearance (C(PAH)) decreased from 1.39 ± 0.27 to 0.98 ± 0.22 ml·min(-1)·g(-1) (P < 0.05) and glomerular filtration rate (GFR) was reduced from 0.50 ± 0.09 to 0.32 ± 0.06 ml·min(-1)·g(-1) (P < 0.05) in NCX1(+/-) mice. In contrast, the NCX1(sm-/-) did not exhibit significant reductions in either C(PAH) (1.16 ± 0.30 to 1.22 ± 0.34 ml·min(-1)·g(-1), P > 0.05) or GFR (0.48 ± 0.08 to 0.41 ± 0.05 ml·min(-1)·g(-1), P > 0.05) during acute ANG II infusions. Using flometry to measure renal blood flow continuously, NCX1(sm-/-) mice had significantly attenuated responses to ANG II infusions (-34.2 ± 3.9%, P < 0.05) compared with those in NCX1(+/-) mice (-48 ± 2%) or in wild-type mice (-69 ± 7%). These data indicate that renal vascular responses to ANG II are attenuated in NCX1(sm-/-) mice compared with NCX1(+/-) mice and that NCX1 contributes to the renal vasoconstriction response to acute ANG II infusions.     

Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II

The present study examined the effects of ANG II on the renal synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and its contribution to the renal vasoconstrictor and the acute and chronic pressor effects of ANG II in rats. ANG II (10(-11) to 10(-7) mol/l) reduced the diameter of renal interlobular arteries treated with inhibitors of nitric oxide synthase and cyclooxygenase, lipoxygenase, and epoxygenase by 81 +/- 8%. Subsequent blockade of the synthesis of 20-HETE with 17-octadecynoic acid (1 micromol/l) increased the ED(50) for ANG II-induced constriction by a factor of 15 and diminished the maximal response by 61%. Graded intravenous infusion of ANG II (5-200 ng/min) dose dependently increased mean arterial pressure (MAP) in thiobutylbarbitol-anesthetized rats by 35 mmHg. Acute blockade of the formation of 20-HETE with dibromododecenyl methylsulfimide (DDMS; 10 mg/kg) attenuated the pressor response to ANG II by 40%. An intravenous infusion of ANG II (50 ng. kg(-1). min(-1)) in rats for 5 days increased the formation of 20-HETE and epoxyeicosatrienoic acids (EETs) in renal cortical microsomes by 60 and 400%, respectively, and increased MAP by 78 mmHg. Chronic blockade of the synthesis of 20-HETE with intravenous infusion of DDMS (1 mg. kg(-1). h(-1)) or EETs and 20-HETE with 1-aminobenzotriazole (ABT; 2.2 mg. kg(-1). h(-1)) attenuated the ANG II-induced rise in MAP by 40%. Control urinary excretion of 20-HETE averaged 350 +/- 23 ng/day and increased to 1,020 +/- 105 ng/day in rats infused with ANG II (50 ng. kg(-1). min(-1)) for 5 days. In contrast, urinary excretion of 20-HETE only rose to 400 +/- 40 and 600 +/- 25 ng/day in rats chronically treated with ANG II and ABT or DDMS respectively. These results suggest that acute and chronic elevations in circulating ANG II levels increase the formation of 20-HETE in the kidney and peripheral vasculature and that 20-HETE contributes to the acute and chronic pressor effects of ANG II.     

Angiotensin II attenuates the natriuresis of water immersion in humans

The hypothesis was tested that suppression of generation of ANG II is one of the mechanisms of the water immersion (WI)-induced natriuresis in humans. In one protocol, eight healthy young males were subjected to 3 h of 1) WI (WI + placebo), 2) WI combined with ANG II infusion of 0.5 ng. kg(-1). min(-1) (WI + ANG II-low), and 3) a seated time control (Con). In another almost identical protocol, 7-10 healthy young males were investigated to delineate the tubular site(s) of action of ANG II by the lithium clearance method (C(Li)) and were on an additional fourth study day subjected to infusion of ANG II at a rate of 1.5 ng. kg(-1). min(-1) (WI + ANG II-high). During WI + placebo, plasma concentration of ANG II decreased from 16 +/- 2 to 8 +/- 1 pg/ml (P < 0.05) and renal sodium excretion increased from 104 +/- 15 to 294 +/- 27 micromol/min (P < 0.05). During WI + ANG II-low, plasma ANG II was not suppressed by WI, and the natriuresis was blunted by 52 +/- 13% (P < 0.05). During WI + ANG II-low and WI + ANG II-high, an increase in C(Li) was prevented that was otherwise observed during WI, and fractional distal reabsorption of sodium was facilitated. In conclusion, maintaining plasma concentration of ANG II unchanged at the level of control attenuates the natriuresis of WI considerably in humans. Therefore, suppression of generation of ANG II is an important mechanism of the natriuresis of WI in humans. Furthermore, infusion of ANG II during WI prevents an otherwise induced increase in C(Li) and facilitates the fractional distal reabsorption of sodium, probably via an effect on aldosterone release.     

Superoxide mediates acute renal vasoconstriction produced by angiotensin II and catecholamines by a mechanism independent of nitric oxide

NAD(P)H oxidases (NOX) and reactive oxygen species (ROS) are involved in vasoconstriction and vascular remodeling during hypertension produced by chronic angiotensin II (ANG II) infusion. These effects are thought to be mediated largely through superoxide anion (O(2)(-)) scavenging of nitric oxide (NO). Little is known about the role of ROS in acute vasoconstrictor responses to agonists. We investigated renal blood flow (RBF) reactivity to ANG II (4 ng), norepinephrine (NE, 20 ng), and alpha(1)-adrenergic agonist phenylephrine (PE, 200 ng) injected into the renal artery (ira) of anesthetized Sprague-Dawley rats. The NOX inhibitor apocynin (1-4 mg.kg(-1).min(-1) ira, 2 min) or the superoxide dismutase mimetic Tempol (1.5-5 mg.kg(-1).min(-1) ira, 2 min) rapidly increased resting RBF by 8 +/- 1% (P < 0.001) or 3 +/- 1% (P < 0.05), respectively. During NO synthase (NOS) inhibition (N(omega)-nitro-l-arginine methyl ester, 25 mg/kg iv), the vasodilation tended to increase (apocynin 13 +/- 4%, Tempol 10 +/- 1%). During control conditions, both ANG II and NE reduced RBF by 24 +/- 4%. Apocynin dose dependently reduced the constriction by up to 44% (P < 0.05). Similarly, Tempol blocked the acute actions of ANG II and NE by up to 48-49% (P < 0.05). In other animals, apocynin (4 mg.kg(-1).min(-1) ira) attenuated vasoconstriction to ANG II, NE, and PE by 46-62% (P < 0.01). During NOS inhibition, apocynin reduced the reactivity to ANG II and NE by 60-72% (P < 0.01), and Tempol reduced it by 58-66% (P < 0.001). We conclude that NOX-derived ROS substantially contribute to basal RBF as well as to signaling of acute renal vasoconstrictor responses to ANG II, NE, and PE in normal rats. These effects are due to O(2)(-) rather than H(2)O(2), occur rapidly, and are independent of scavenging of NO.     

ANG II-induced downregulation of RBF after a prolonged reduction of renal perfusion pressure is due to pre- and postglomerular constriction

Previous experiments from our laboratory showed that longer-lasting reductions in renal perfusion pressure (RPP) are associated with a gradual decrease in renal blood flow (RBF) that can be abolished by clamping plasma ANG II concentration ([ANG II]). The aim of the present study was to investigate the mechanisms behind the RBF downregulation in halothane-anesthetized Sprague-Dawley rats during a 30-min reduction in RPP to 88 mmHg. During the 30 min of reduced RPP we also measured glomerular filtration rate (GFR), proximal tubular pressure (P(prox)), and proximal tubular flow rate (Q(LP)). Early distal tubular fluid conductivity was measured as an estimate of early distal [NaCl] ([NaCl](ED)), and changes in plasma renin concentration (PRC) over time were measured. During 30 min of reduced RPP, RBF decreased gradually from 6.5 +/- 0.3 to 6.0 +/- 0.3 ml/min after 5 min (NS) to 5.2 +/- 0.2 ml/min after 30 min (P < 0.05). This decrease occurred in parallel with a gradual increase in PRC from 38.2 +/- 11.0 x 10(-5) to 87.1 +/- 25.1 x 10(-5) Goldblatt units (GU)/ml after 5 min (P < 0.05) to 158.5 +/- 42.9 x 10(-5) GU/ml after 30 min (P < 0.01). GFR, P(prox), and [NaCl](ED) all decreased significantly after 5 min and remained low. Estimates of pre- and postglomerular resistances showed that the autoregulatory mechanisms initially dilated preglomerular vessels to maintain RBF and GFR. However, after 30 min of reduced RPP, both pre- and postglomerular resistance had increased. We conclude that the decrease in RBF over time is caused by increases in both pre- and postglomerular resistance due to rising plasma renin and ANG II concentrations.     

p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats

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

Angiotensin-induced defects in renal oxygenation: role of oxidative stress

We tested the hypothesis that superoxide anion (O(2)(-).) generated in the kidney by prolonged angiotensin II (ANG II) reduces renal cortical Po(2) and the use of O(2) for tubular sodium transport (T(Na):Q(O(2))). Groups (n = 8-11) of rats received angiotensin II (ANG II, 200 ng.kg(-1).min(-1) sc) or vehicle for 2 wk with concurrent infusions of a permeant nitroxide SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, 200 nmol.kg(-1).min(-1)) or vehicle. Rats were studied under anesthesia with measurements of renal oxygen usage and Po(2) in the cortex and tubules with a glass electrode. Compared with vehicle, ANG II increased mean arterial pressure (107 +/- 4 vs. 146 +/- 6 mmHg; P < 0.001), renal vascular resistance (42 +/- 3 vs. 65 +/- 7 mmHg.ml(-1).min(-1).100 g(-1); P < 0.001), renal cortical NADPH oxidase activity (2.3 +/- 0.2 vs. 3.6 +/- 0.4 nmol O(2)(-)..min(-1).mg(-1) protein; P < 0.05), mRNA and protein expression for p22(phox) (2.1- and 1.8-fold respectively; P < 0.05) and reduced the mRNA for extracellular (EC)-SOD (-1.8 fold; P < 0.05). ANG II reduced the Po(2) in the proximal tubule (39 +/- 1 vs. 34 +/- 2 mmHg; P < 0.05) and throughout the cortex and reduced the T(Na):Q(O(2)) (17 +/- 1 vs. 9 +/- 2 mumol/mumol; P < 0.001). Tempol blunted or prevented all these effects of ANG II. The effects of prolonged ANG II to cause hypertension, renal vasoconstriction, renal cortical hypoxia, and reduced efficiency of O(2) usage for Na(+) transport, activation of NADPH oxidase, increased expression of p22(phox), and reduced expression of EC-SOD can be ascribed to O(2)(-). generation because they are prevented by an SOD mimetic.     

Role of NOX2 in the regulation of afferent arteriole responsiveness

NADPH oxidases (NOX) are the major source of reactive oxygen species (ROS) in the vasculature and contribute to the control of renal perfusion. The role of NOX2 in the regulation of blood pressure and afferent arteriole responsiveness was investigated in NOX2(-/-) and wild-type mice. Arteriole constrictions to ANG II (10(-14)-10(-6) mol/l) were weaker in NOX2(-/-) compared with wild types. N(omega)-nitro-l-arginine methyl ester (l-NAME; 10(-4) mol/l) treatment reduced basal diameters significantly more in NOX2(-/-) (-18%) than in wild types (-6%) and augmented ANG II responses. Adenosine (10(-11)-10(-4) mol/l) constricted arterioles of wild types but not of NOX2(-/-). However, simultaneous inhibition of adenosine type-2 receptors induced vasoconstriction, which was stronger in NOX2(-/-). Adenosine (10(-8) mol/l) enhanced the ANG II response in wild type, but not in NOX2(-/-). This sensitizing effect by adenosine was abolished by apocynin. Chronic ANG II pretreatment (14 days) did not change the ANG II responses in NOX2(-/-), but strengthened the response in wild types. ANG II pretreatment augmented the l-NAME response in NOX2(-/-) (-33%), but not in wild types. Simultaneous application of l-NAME and ANG II caused a stronger constriction in the NOX2(-/-) (-64%) than in wild types (-46%). Basal blood pressures were similar in both genotypes, however, chronic ANG II infusion elevated blood pressure to a greater extent in wild-type (15 +/- 1%) than in NOX2(-/-) (8 +/- 1%) mice. In conclusion, NOX2 plays an important role in the control of afferent arteriole tone and is involved in the contractile responses to ANG II and/or adenosine. NOX2 can be activated by elevated ANG II and may play an important role in ANG II-induced hypertension. NOX2-derived ROS scavenges nitric oxide, causing subsequent nitric oxide-deficiency.     

Predominant postglomerular vascular resistance response to reflex renal sympathetic nerve activation during ANG II clamp in rabbits

We have shown previously that a moderate reflex increase in renal sympathetic nerve activity (RSNA) elevated glomerular capillary pressure, whereas a more severe increase in RSNA decreased glomerular capillary pressure. This suggested that the nerves innervating the glomerular afferent and efferent arterioles could be selectively activated, allowing differential control of glomerular capillary pressure. A caveat to this conclusion was that intrarenal actions of neurally stimulated ANG II might have contributed to the increase in postglomerular resistance. This has now been investigated. Anesthetized rabbits were prepared for renal micropuncture and RSNA recording. One group (ANG II clamp) received an infusion of an angiotensin-converting enzyme inhibitor (enalaprilat, 2 mg/kg bolus plus 2 mg.kg(-1).h(-1)) plus ANG II ( approximately 20 ng.kg(-1).min(-1)), the other vehicle. Measurements were made before (room air) and during 14% O(2). Renal blood flow decreased less during ANG II clamp compared with vehicle [9 +/- 1% vs. 20 +/- 4%, interaction term (P(GT)) < 0.05], despite a similar increase in RSNA in response to 14% O(2) in the two groups. Arterial pressure and glomerular filtration rate were unaffected by 14% O(2) in both groups. Glomerular capillary pressure increased from 33 +/- 1 to 37 +/- 1 mmHg during ANG II clamp and from 33 +/- 2 to 35 +/- 1 mmHg in the vehicle group before and during 14% O(2), respectively (P(GT) < 0.05). During ANG II clamp, postglomerular vascular resistance was still increased in response to RSNA during 14% O(2), demonstrating that the action of the renal nerves on the postglomerular vasculature was independent of the renin-angiotensin system. This further supports our hypothesis that increases in RSNA can selectively control pre- and postglomerular vascular resistance and therefore glomerular ultrafiltration.     

Enhanced renal expression of preproendothelin mRNA during chronic angiotensin II hypertension

The purpose of this study was to determine the role of endothelin in mediating the renal hemodynamic and arterial pressure changes observed during chronic ANG II-induced hypertension. ANG II (50 ng x kg(-1) x min(-1)) was chronically infused into the jugular vein by miniosmotic pump for 2 wk in male Sprague-Dawley rats with and without endothelin type A (ET(A))-receptor antagonist ABT-627 (5 mg x kg(-1) x day(-1)) pretreatment. Arterial pressure increased in ANG II rats compared with control rats (149 +/- 5 vs. 121 +/- 6 mmHg, P < 0.05, respectively). Renal expression of preproendothelin mRNA was increased by approximately 50% in both the medulla and cortex of ANG II rats. The hypertensive effect of ANG II was completely abolished in rats pretreated with the ET(A)-receptor antagonist (114 +/- 5 mmHg, P < 0.05). Glomerular filtration rate was decreased by 33% in ANG II rats, and this response was attenuated in rats pretreated with ET(A)-receptor antagonist. These data indicate that activation of the renal endothelin system by ANG II may play an important role in mediating chronic renal and hypertensive actions of ANG II.     

Differential ANG II generation in plasma and tissue of mice with decreased expression of the ACE gene

We utilized mice with homozygous disruption of angiotensin-converting enzyme (ACE) (-/-), mice with heterozygous deletion of ACE (+/-), and wild-type mice (+/+) to test the hypothesis that genetic variation in ACE modulates tissue and plasma angiotensin (ANG) II concentrations. With the use of ANG I as substrate, kidney, heart, and lung ACE activity was reduced 80% in -/- mice compared with +/+ mice. However, ANG II concentrations and ANG II-to-ANG I ratios in the kidney, heart, and lung did not differ among genotypes. In contrast, plasma ANG II concentrations in -/- mice were <2 fmol/ml, whereas plasma ANG I concentrations were extremely high (765 fmol/ml). Chymase activity was increased 14-fold in the kidney (P < 0.05) and 1.5-fold in the heart (P < 0.05) of -/- versus +/+ mice but did not differ among genotypes in the lung. ANG II formation from enzymes other than ACE and chymase contributed <2% of total ANG II formation in all genotypes. These data suggest that ACE is essential to ANG II formation in the vascular space, whereas chymase may provide an important mechanism in maintaining steady-state ANG II levels in tissue.     

Renal medullary nitric oxide deficit of Dahl S rats enhances hypertensive actions of angiotensin II

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng. kg(-1). min(-1)) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 +/- 8 to 117 +/- 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng. kg(-1). min(-1) did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 +/- 2 to 138 +/- 2 mmHg (P < 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of L-arginine (300 microg. kg(-1). min(-1)) into the renal medulla of Dahl S rats. Intravenous infusion of L-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.     

Effects of angiotensin II on regional afferent and efferent arteriole dimensions and the glomerular pole

The diversity of renal arteriole diameters in different cortical regions has important consequences for control of glomerular capillary pressure. We examined whether intrarenal angiotensin II (ANG II; 0.1, 1, or 5 ng. kg(-1). min(-1)) in anesthetized rabbits acts preferentially on pre- or postglomerular vessels using vascular casting. ANG II produced dose-related reductions in afferent and efferent diameters in the outer, mid, and inner cortex, without effecting arterial pressure. Afferent diameter decreased more than efferent in the outer and mid cortex (P < 0.05) but by a similar extent in juxtamedullary nephrons (P = 0.58). Calculated efferent resistance increased more than afferent, especially in the outer cortex (127 vs. 24 units; 5 ng. kg(-1). min(-1) ANG II). ANG II produced significant dose-related increases in the distance between the arterioles at the entrance to the glomerular pole in all regions. Thus afferent diameter decreased more in response to ANG II, but efferent resistance rose more due to smaller resting luminal dimensions. The results also indicate that glomerular pole dimensions change in response to ANG II.     

Drinking behavior elicited by central injection of angiotensin II: roles for protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.     

Angiotensin II hypertension is attenuated in interleukin-6 knockout mice

Plasma levels of IL-6 correlate with high blood pressure under many circumstances, and ANG II has been shown to stimulate IL-6 production from various cell types. This study tested the role of IL-6 in mediating the hypertension caused by high-dose ANG II and a high-salt diet. Male C57BL6 and IL-6 knockout (IL-6 KO) mice were implanted with biotelemetry devices and placed in metabolic cages to measure mean arterial pressure (MAP), heart rate (HR), sodium balance, and urinary albumin excretion. Baseline MAP during the control period averaged 114 +/- 1 and 109 +/- 1 mmHg for wild-type (WT) and IL-6 KO mice, respectively, and did not change significantly when the mice were placed on a high-salt diet (HS; 4% NaCl). ANG II (90 ng/min sc) caused a rapid increase in MAP in both groups, to 141 +/- 9 and 141 +/- 4 in WT and KO mice, respectively, on day 2. MAP plateaued at this level in KO mice (134 +/- 2 mmHg on day 14 of ANG II) but began to increase further in WT mice by day 4, reaching an average of 160 +/- 4 mmHg from days 10 to 14 of ANG II. Urinary albumin excretion on day 4 of ANG II was not different between groups (9.18 +/- 4.34 and 8.53 +/- 2.85 microg/2 days for WT and KO mice). By day 14, albumin excretion was nearly fourfold greater in WT mice, but MAP dropped rapidly back to control levels in both groups when the ANG II was stopped after 14 days. Thus the approximately 30 mmHg greater ANG II hypertension in the WT mice suggests that IL-6 contributes significantly to ANG II-salt hypertension. In addition, the early separation in MAP, the albumin excretion data, and the rapid, post-ANG II recovery of MAP suggest an IL-6-dependent mechanism that is independent of renal injury.     

Modulation of V1-receptor-mediated renal vasoconstriction by epoxyeicosatrienoic acids

This study examined the effects of renal arterial infusion of a selective cytochrome P-450 epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 2 mg/kg plus 1.5 mg.kg(-1).h(-1)), on renal hemodynamic responses to infusions of [Phe(2),Ile(3),Orn(8)]vasopressin and ANG II into the renal artery of anesthetized rabbits. MS-PPOH did not affect basal renal blood flow (RBF) or cortical or medullary blood flow measured by laser-Doppler flowmetry (CLDF/MLDF). In vehicle-treated rabbits, [Phe(2),Ile(3),Orn(8)]vasopressin (30 ng.kg(-1).min(-1)) reduced MLDF by 62 +/- 7% but CLDF and RBF were unaltered. In MS-PPOH-treated rabbits, RBF and CLDF fell by 51 +/- 8 and 59 +/- 13%, respectively, when [Phe(2),Ile(3),Orn(8)]vasopressin was infused. MS-PPOH had no significant effects on the MLDF response to [Phe(2),Ile(3),Orn(8)]vasopressin (43 +/- 9% reduction). ANG II (20 ng.kg(-1).min(-1)) reduced RBF by 45 +/- 10% and CLDF by 41 +/- 14%, but MLDF was not significantly altered. MS-PPOH did not affect blood flow responses to ANG II. Formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DiHETEs) was 49% lower in homogenates prepared from the renal cortex of MS-PPOH-treated rabbits than from vehicle-treated rabbits. MS-PPOH had no effect on the renal formation of 20-hydroxyeicosatetraenoic acid (20-HETE). Incubation of renal cortical homogenates from untreated rabbits with [Phe(2),Ile(3),Orn(8)]vasopressin (0.2-20 ng/ml) did not affect formation of EETs, DiHETEs, or 20-HETE. These results do not support a role for de novo EET synthesis in modulating renal hemodynamic responses to ANG II. However, EETs appear to selectively oppose V(1)-receptor-mediated vasoconstriction in the renal cortex but not in the medullary circulation and contribute to the relative insensitivity of cortical blood flow to V(1)-receptor activation [corrected].     

Volume expansion during NOS substrate donation with L-arginine: regulatory offsetting of renal response?

The responses to infusion of nitric oxide synthase substrate (L-arginine 3 mg.kg(-1).min(-1)) and to slow volume expansion (saline 35 ml/kg for 90 min) alone and in combination were investigated in separate experiments. L-Arginine left blood pressure and plasma ANG II unaffected but decreased heart rate (6 +/- 2 beats/min) and urine osmolality, increased glomerular filtration rate (GFR) transiently, and caused sustained increases in sodium excretion (fourfold) and urine flow (0.2 +/- 0.0 to 0.7 +/- 0.1 ml/min). Volume expansion increased arterial blood pressure (102 +/- 3 to 114 +/- 3 mmHg), elevated GFR persistently by 24%, and enhanced sodium excretion to a peak of 251 +/- 31 micromol/min, together with marked increases in urine flow, osmolar and free water clearances, whereas plasma ANG II decreased (8.1 +/- 1.7 to 1.6 +/- 0.3 pg/ml). Combined volume expansion and L-arginine infusion tended to increase arterial blood pressure and increased GFR by 31%, whereas peak sodium excretion was enhanced to 335 +/- 23 micromol/min at plasma ANG II levels of 3.0 +/- 1.1 pg/ml; urine flow and osmolar clearance were increased at constant free water clearance. In conclusion, L-arginine 1) increases sodium excretion, 2) decreases basal urine osmolality, 3) exaggerates the natriuretic response to volume expansion by an average of 50% without persistent changes in GFR, and 4) abolishes the increase in free water clearance normally occurring during volume expansion. Thus L-arginine is a natriuretic substance compatible with a role of nitric oxide in sodium homeostasis, possibly by offsetting/shifting the renal response to sodium excess.     

Effects of cardiac hormones on arterial pressure and sodium excretion in NPRA knockout mice

These studies were designed to determine if the atria contains natriuretic substances that act through a non-natriuretic peptide type A (NPRA) receptor mechanism. C57BL/6 mice, either wild-type NPRA++ (WT) or NPRA-- knockout (KO), were anesthetized with pentobarbital. Catheters were placed in the trachea, carotid artery, jugular vein, and bladder. Urine was collected for six 30-min periods. Both groups received an iv injection of 100 ng of rat atrial natriuretic peptide (rANP) in 200 microl of saline after the first period (30 mins) and 200 microl of rat atrial extract after the fourth period (120 mins). ANP injection increased urine flow (UF) to 2.7 +/- 0.5 microl/min in the WT versus 1.9 +/- 0.2 in KO. Extract increased UF to 7.9 +/- 1.5 microl/min in WT versus 2.7 +/- 0.4 in KO (P < 0.01). ANP increased sodium excretion (ENa) to 0.47 +/- 0.10 micromoles/min in WT versus 0.27 +/- 0.04 in KO (P < 0.05). Extract increased ENa to 1.44 +/- 0.47 micromoles/min in WT versus 0.26 +/- 0.06 in KO (P < 0.05). Extract decreased mean arterial pressure (MAP) to 62 +/- 3 mm Hg in the WT versus 81 +/- 5 in KO (P < 0.01). ENa and MAP responses to extract in KO were not different from responses to 200 microl of saline. A constant 150-min infusion of rat atrial extract increased urine flow by 3-fold and ENa by 5-fold (both P < 0.05) in the WT mice but had no significant effect in the KO mice. Thus, acute renal and MAP responses to atrial extracts require the NPRA receptor.