Tumor necrosis factor-α, kidney function, and hypertension.

Hypertension is considered to be a low-grade inflammatory condition characterized by the presence of various proinflammatory cytokines. Tumor necrosis factor-α (TNF-α) is a constituent of the proinflammatory cytokines that is associated with salt-sensitive hypertension (SSH) and related renal injury. Elevated angiotensin II (ANG II) and other factors such as oxidative stress conditions promote TNF-α formation. Many recent studies have provided evidence that TNF-α exerts a direct renal action by regulating hemodynamic and excretory function in the kidney. The cytokine incites a strong natriuretic response and plays a part in regulation of the intrarenal renin-angiotensin system. The exact mechanistic role of TNF-α in the development of SSH is as yet poorly understood. While TNF-α antagonism has been shown to attenuate hypertensive responses in many hypertensive animal models, contrasting findings demonstrate that the direct systemic administration of TNF-α usually induces hypotensive as well as natriuretic responses, indicating a counterregulatory role of TNF-α in SSH. Differential activities of two cell surface receptors of TNF-α (receptor type 1 and type 2) may explain the contradictory functions of TNF-α in the setting of hypertension. This short review will evaluate ongoing research studies that investigate the action of TNF-α within the kidney and its role as an influential pathophysiological variable in the development of SSH and renal injury. This information may help to develop specific TNF-α receptor targeting as an effective treatment strategy in this clinical condition.

Augmentation of Nitric Oxide Deficient Hypertension by High Salt Diet Is Associated With Reduced TNF-α Receptor Type 1 Expression in the Kidneys.

BACKGROUND: High salt (HS) intake induces an augmented hypertensive response to nitric oxide (NO) inhibition, though it causes minimal changes in blood pressure (BP) in NO intact condition. The cause of such augmentation is not known. HS induces tumor necrosis factor-alpha (TNFα) production that causes natriuresis via activation of its receptor type 1 (TNFR1). We hypothesized that NO deficiency reduces renal TNFR1 activity, leading to enhanced sodium retention and hypertension. METHODS: We examined the changes in renal TNFR1 protein expression (Immunohistochemistry analyses) after HS (4% NaCl) intake in wild-type mice (WT, C57BL6) treated with a NO synthase (NOS) inhibitor, nitro-l-arginine methyl ester (L-NAME; 0.05 mg/min/g; osmotic mini-pump), as well as in endothelial NOS knockout mice (eNOSKO) and compared the responses in WT mice with normal salt (NS; 0.3% NaCl) intake. BP was measured with tail-cuff plethysmography and 24-hour urine collections were made using metabolic cages. RESULTS: HS alone did not alter mean BP in untreated mice (76 ±â€…3 to 77 ±â€…1 mm Hg) but induced an augmented response in L-NAME treated (106 ±â€…1 vs. 97 ±â€…2 mm Hg) and in eNOSKO (107 ±â€…2 vs. 89 ±â€…3 mm Hg) mice. The percentage area of TNFR1 expression in renal tissue was higher in WT + HS (4.1 + 0.5%) than in WT + NS mice (2.7 ±â€…0.6%). However, TNFR1 expression was significantly lower in L-NAME treated WT + NS (0.9 ±â€…0.1%) and in eNOSKO + NS (1.4 ±â€…0.2%) than in both WT + NS and WT + HS mice. CONCLUSIONS: These data indicate that TNFR1 activity is downregulated in NO deficient conditions, which facilitates salt retention leading to augmented hypertension during HS intake.

Protective role of the endothelial isoform of nitric oxide synthase in ANG II-induced inflammatory responses in the kidney.

In the present study, we examine the hypothesis that the nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a protective role in the development of ANG II-induced hypertension and renal injury by minimizing oxidative stress and the inflammation induced by TNF-α. Systolic blood pressure (SBP) and renal injury responses to chronic infusions of ANG II (via implanted minipumps) were evaluated for 2 wk in wild-type (WT) and in eNOS knockout mice (KO) cotreated with or without a superoxide (O2(-)) scavenger, tempol (400 mg/l in the drinking water), or a TNF-α receptor blocker, etanercept (5 mg/kg/day ip). In study 1, when ANG II was given at a dose of 25 ng/min, it increased mean SBP in WT mice (Δ36 ± 3 mmHg; n = 7), and this effect was attenuated in mice pretreated with tempol (Δ24 ± 3 mmHg; n = 6). In KO mice (n = 9), this dose of ANG II resulted in severe renal injury associated with high mortality. To avoid this high mortality in KO, study 2 was conducted with a lower dose of ANG II (10 ng/min) that increased SBP slightly in WT (Δ17 ± 7 mmHg; n = 6) but exaggeratedly in KO (Δ48 ± 12 mmHg, n = 6) associated with severe renal injury. Cotreatment with either tempol (n = 6) or etanercept (n = 6) ameliorated the hypertensive, as well as the renal injury responses in KO compared with WT. These data demonstrate a protective role for eNOS activity in preventing renal inflammatory injury and hypertension induced by chronic increases in ANG II.

TNF-α type 2 receptor mediates renal inflammatory response to chronic angiotensin II administration with high salt intake in mice.

Tumor necrosis factor-alpha (TNF-α) has been implicated in salt-sensitive hypertension and renal injury (RI) induced by angiotensin II (ANG II). To determine the receptor type of TNF-α involved in this mechanism, we evaluated the responses to chronic ANG II infusion (25 ng/min by implanted minipump) given with high-salt diet (HS; 4% NaCl) for 2 wk in gene knockout mice for TNF-α receptor type 1 (TNFR1KO; n = 6) and type 2 (TNFR2KO; n = 6) and compared the responses with those in wild-type (WT; C57BL/6; n = 6) mice. Blood pressure in these mice was measured by implanted radiotelemetry as well as by tail-cuff plethysmography. RI responses were assessed by measuring macrophage cell infiltration (CD68(+) immunohistochemistry), glomerulosclerosis (PAS staining), and interstitial fibrosis (Gomori's trichrome staining) in renal tissues at the end of the treatment period. The increase in mean arterial pressure induced by ANG II + HS treatment was not different in these three groups of mice (TNFR1KO, 114 ± 1 to 161 ± 7 mmHg; TNFR2KO, 113 ± 1 to 161 ± 3 mmHg; WT, 110 ± 3 to 154 ± 3 mmHg). ANG II + HS-induced RI changes were similar in TNFR1KO mice but significantly less in TNFR2KO mice (macrophage infiltration, 0.02 ± 0.01 vs. 1.65 ± 0.45 cells/mm(2); glomerulosclerosis, 26.3 ± 2.6 vs. 35.7 ± 2.2% area; and interstitial fibrosis, 5.2 ± 0.6 vs. 8.1 ± 1.1% area) compared with the RI changes in WT mice. The results suggest that a direct activation of TNF-α receptors may not be required in inducing hypertensive response to chronic ANG II administration with HS intake, but the induction of inflammatory responses leading to renal injury are mainly mediated by TNF-α receptor type 2.

Tumor necrosis factor-α receptor type 1, not type 2, mediates its acute responses in the kidney.

Acute administration of tumor necrosis factor-α (TNF-α) resulted in decreases in renal blood flow (RBF) and glomerular filtration rate (GFR) but induced diuretic and natriuretic responses in mice. To define the receptor subtypes involved in these renal responses, experiments were conducted to assess the responses to human recombinant TNF-α (0.3 ng·min(-1)·g body wt(-1) iv infusion for 75 min) in gene knockout (KO) mice for TNF-α receptor type 1 (TNFαR1 KO, n = 5) or type 2 (TNFαR2 KO, n = 6), and the results were compared with those obtained in corresponding wild-type [WT (C57BL/6), n = 6] mice. Basal levels of RBF (PAH clearance) and GFR (inulin clearance) were similar in TNFαR1 KO, but were lower in TNFαR2 KO, than WT mice. TNF-α infusion in WT mice decreased RBF and GFR but caused a natriuretic response, as reported previously. In TNFαR1 KO mice, TNF-α infusion failed to cause such vasoconstrictor or natriuretic responses; rather, there was an increase in RBF and a decrease in renal vascular resistance. Similar responses were also observed with infusion of murine recombinant TNF-α in TNFαR1 KO mice (n = 5). However, TNF-α infusion in TNFαR2 KO mice caused changes in renal parameters qualitatively similar to those observed in WT mice. Immunohistochemical analysis in kidney slices from WT mice demonstrated that while both receptor types were generally located in the renal vascular and tubular cells, only TNFαR1 was located in vascular smooth muscle cells. There was an increase in TNFαR1 immunoreactivity in TNFαR2 KO mice, and vice versa, compared with WT mice. Collectively, these functional and immunohistological findings in the present study demonstrate that the activation of TNFαR1, not TNFαR2, is mainly involved in mediating the acute renal vasoconstrictor and natriuretic actions of TNF-α.

AT1 receptor-mediated augmentation of angiotensinogen, oxidative stress, and inflammation in ANG II-salt hypertension.

Augmentation of intrarenal angiotensinogen (AGT) synthesis, secretion, and excretion is associated with the development of hypertension, renal oxidative stress, and tissue injury during ANG II-dependent hypertension. High salt (HS) exacerbates hypertension and kidney injury, but the mechanisms remain unclear. In this study, we determined the consequences of HS intake alone compared with chronic ANG II infusion and combined HS plus ANG II on the stimulation of urinary AGT (uAGT), renal oxidative stress, and renal injury markers. Sprague-Dawley rats were subjected to 1) a normal-salt diet [NS, n = 5]; 2) HS diet [8% NaCl, n = 5]; 3) ANG II infusion in NS rats [ANG II 80 ng/min, n = 5]; 4) ANG II infusion in HS rats [ANG II+HS, n = 5]; and 5) ANG II infusion in HS rats treated with ANG II type 1 receptor blocker (ARB) [ANG II+HS+ARB, n = 5] for 14 days. Rats fed a HS diet alone did not show changes in systolic blood pressure (SBP), proteinuria, cell proliferation, or uAGT excretion although they did exhibit mesangial expansion, collagen deposition, and had increased NADPH oxidase activity accompanied by increased peroxynitrite formation in the kidneys. Compared with ANG II rats, the combination of ANG II infusion and a HS diet led to exacerbation in SBP (175 ± 10 vs. 221 ± 8 mmHg; P < 0.05), proteinuria (46 ± 7 vs. 127 ± 7 mg/day; P < 0.05), and uAGT (1,109 ± 70 vs.. 7,200 ± 614 ng/day; P < 0.05) associated with greater collagen deposition, mesangial expansion, interstitial cell proliferation, and macrophage infiltration. In both ANG II groups, the O(2)(-) levels were increased due to increased NADPH oxidase activity without concomitant increases in peroxynitrite formation. The responses in ANG II rats were prevented or ameliorated by ARB treatment. The results indicate that HS independently stimulates ROS formation, which may synergize with the effect of ANG II to limit peroxynitrite formation, leading to exacerbation of uAGT and greater injury during ANG II salt hypertension.

Angiotensin II-induced natriuresis is attenuated in knockout mice lacking the receptors for tumor necrosis factor-α.

Intravenous infusion of relatively higher doses of angiotensin II (AngII) elicits natriuresis as opposed to its usual anti-natruretic response. As AngII can induce tumor necrosis factor-α (TNFα) production which elicits natriuresis via its action on TNFα receptor type 1 (TNFR1), we hypothesize that the concomitant release of TNFα contributes to the natriuretic response to AngII. Responses to AngII infusion (1 ng min-1  g-1 for 75 min, iv) were evaluated in anesthetized knockout (KO) mice lacking TNFR1 (n = 6) and TNFR2 (TNFα receptor type 2; n = 6) and compared these responses with those in wild type (WT; n = 6) mice. Arterial pressure (AP) was recorded from a cannula placed in the carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances, respectively. Urine was collected from a catheter placed in the bladder. AngII caused similar increases (p < 0.05 vs basal values) in AP (WT, 37 ± 5%; TNFR1KO, 35 ± 4%; TNFR2KO, 30 ± 4%) and decreases (p < 0.05) in RBF (WT, -39 ± 5%; TNFR1KO, -28 ± 6%; TNFR2KO, -31 ± 4%) without significant changes in GFR (WT, -17 ± 7%; TNFR1KO, -18 ± 7%; TNFR2KO, -12 ± 7%). However, despite similar changes in AP and renal hemodynamics, AngII induced increases (p < 0.05) in urinary sodium excretion in WT (3916 ± 942%) were less in the KO strains, more or less in TNFR1KO (473 ± 170%) than in TNFR2KO (1176 ± 168%). These data indicate that TNF-α receptors, particularly TNFR1 are involved in the natriuretic response that occur during acute infusion of AngII and thus, plays a protective role in preventing excessive salt retention at clinical conditions associated with elevated AngII level.

High-salt intake reduces renal tissue levels of inflammatory cytokines in mice.

High salt (HS) intake is usually considered as an aggravating factor to induce inflammatory renal injury. However, the changes in the renal levels of inflammatory cytokines during HS intake is not yet clearly defined. We hypothesize that HS increases renal levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) but decreases interleukin-10 (IL-10; anti-inflammatory cytokine) and these responses exacerbate in NO deficient conditions. Both wild-type (WT) and endothelial NO synthase knockout (eNOSKO) mice (~8 weeks old, n = 6 in each group) were given normal-salt (NS; 0.3% NaCl) and HS (4% NaCl) containing diets for 2 weeks. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography and urine collections were made using metabolic cages. Basal SBP was higher in eNOSKO than WT mice (131 ± 7 vs 117 ± 3 mmHg; p < .05). HS intake for 2 weeks increased SBP in eNOSKO (161 ± 5 mmHg) but not in WT mice. In NS groups, the cytokine levels in renal tissues (measured using ELISA kits and expressed in pg/mg protein) were significantly higher in eNOSKO than WT mice (TNF-α, 624 ± 67 vs. 325 ± 73; IL-6, 619 ± 106 vs. 166 ± 61; IL-10, 6,087 ± 567 vs. 3,929 ± 378). Interestingly, these cytokine levels in HS groups were significantly less both in WT (TNF-α, 114 ± 17; IL-6, 81 ± 14; IL-10, 865 ± 130) and eNOSKO (TNF-α, 115 ± 18; IL-6, 56 ± 7; IL-10, 882 ± 141) mice. These findings indicate that HS induces downregulation of cytokines in the kidney. Such HS-induced reduction in cytokines, particularly TNF-α (a natriuretic agent), would facilitate more salt-retention, and thus, leading to salt-sensitive hypertension in NO deficient conditions.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor-alpha (TNF-α) levels increase. However, TNF-α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF-α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild-type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail-cuff plethysmography, and 24-h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

Involvement of tumor necrosis factor-alpha in natriuretic response to systemic infusion of nitric oxide synthase inhibitor in anesthetized mice.

Systemic infusion of TNF-alpha exerts renal vasoconstriction but caused marked natriuresis in mice. Similar renal responses were also observed during systemic infusion of nitric oxide (NO) synthase inhibitors as opposed to their usual antinatriuretic responses when administered intrarenally. In the present study, we examined the hypothesis that acute NO blockade systemically induces TNF-alpha generation. which induces this natriuretic response. Renal responses to intravenous infusion of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 0.2 microg x min(-1) x g body wt(-1) for 85 min) and its impact on the plasma level of TNF-alpha were evaluated in anesthetized mice. Plasma TNF-alpha was undetected in untreated mice (n = 7) but was elevated in L-NAME-treated mice (109 +/- 22 pg/ml; P < 0.01 vs. untreated group; n = 7) along with an increase in TNF-alpha protein expression in kidney tissue. L-NAME infusion caused a usual increase in mean arterial pressure (MAP; 98 +/- 3 to 122 +/- 3 mmHg; P < 0.01) and decreases in renal blood flow (RBF; 8.6 +/- 0.3 to 4.4 +/- 0.2 ml x min(-1) x g(-1); P < 0.01) and glomerular filtration rate (GFR; 1.14 +/- 0.07 to 0.77 +/- 0.04 ml x min(-1) x g(-1); P < 0.01) with a marked increase in sodium excretion (U(Na)V; 0.48 +/- 0.10 to 3.52 +/- 0.85 micromol x min(-1) x g(-1); P < 0.01). Interestingly, in mice (n = 7) pretreated with the TNF-alpha blocker etanercept (5 mg/kg sc), the U(Na)V response to l-NAME infusion was markedly blunted (0.58 +/- 0.08 to 1.22 +/- 0.28 micromol x min(-1) x g(-1); P = NS) although responses for MAP, RBF, and GFR were mostly unchanged. However, pretreatment with the superoxide scavenger tempol in mice (n = 7) did not alter the U(Na)V response to L-NAME. These data demonstrate that L-NAME-induced natriuresis is mediated, at least in part, by concomitant generation of TNF-alpha during NO blockade.

High-salt intake enhances superoxide activity in eNOS knockout mice leading to the development of salt sensitivity.

A deficiency in nitric oxide (NO) generation leads to salt-sensitive hypertension, but the role of increased superoxide (O(2)(-)) in such salt sensitivity has not been delineated. We examined the hypothesis that an enhancement in O(2)(-) activity induced by high-salt (HS) intake under deficient NO production contributes to the development of salt-sensitive hypertension. Endothelial NO synthase knockout (eNOS KO; total n = 64) and wild-type (WT; total n = 58) mice were given diets containing either normal (NS; 0.4%) or high-salt (HS; 4%) for 2 wk. During this period, mice were chronically treated with a O(2)(-) scavenger, tempol (400 mg/l), or an inhibitor of NADPH oxidase, apocynin (1 g/l), in drinking water or left untreated (n = 6-8 per group). Blood pressure was measured by radiotelemetry and 24-h urine samples were collected in metabolic cages. Basal mean arterial pressure (MAP) in eNOS KO was higher (125 +/- 4 vs. 106 +/- 3 mmHg) compared with WT. Feeding HS diet did not alter MAP in WT but increased it in eNOS KO to 166 +/- 9 mmHg. Both tempol and apocynin treatment significantly attenuated the MAP response to HS in eNOS KO (134 +/- 3 and 139 +/- 4 mmHg, respectively). Basal urinary 8-isoprostane excretion rates (U(Iso)V), a marker for endogenous O(2)(-) activity, were similar (2.8 +/- 0.2 and 2.4 +/- 0.3 ng/day) in both eNOS KO and WT mice. However, HS increased U(Iso)V more in eNOS KO than in WT (4.6 +/- 0.3 vs. 3.8 +/- 0.2 ng/day); these were significantly attenuated by both tempol and apocynin treatment. These data indicate that an enhancement in O(2)(-) activity contributes substantially to the development of salt-sensitive hypertension under NO-deficient conditions.

Low Nitric Oxide Bioavailability Increases Renin Production in the Collecting Duct.

In the kidney, the stimulation of renin production by the collecting duct (CD-renin) contributes to the development of hypertension. The CD is a major nephron segment for the synthesis of nitric oxide (NO), and low NO bioavailability in the renal medulla is associated with hypertension. However, it is unknown whether NO regulates renin production in the CD. To test the hypothesis that low intrarenal NO levels stimulate the production of CD-renin, we first examined renin expression in the distal nephron segments of CD-eNOS deficient mice. In these mice, specific CD-renin immunoreactivity was increased compared to wild-type littermates; however, juxtaglomerular (JG) renin was not altered. To further assess the intracellular mechanisms involved, we then treated M-1 cells with either 1 mM L-NAME (L-arginine analog), an inhibitor of NO synthase activity, or 1 mM NONOate, a NO donor. Both treatments increased intracellular renin protein levels in M-1 cells. However, only the inhibition of NOS with L-NAME stimulated renin synthesis and secretion as reflected by the increase in Ren1C transcript and renin protein levels in the extracellular media, respectively. In addition, NONOate induced a fast mobilization of cGMP and intracellular renin accumulation. These response was partially prevented by guanylyl cyclase inhibition with ODQ (1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1]. Accumulation of intracellular renin was blocked by protein kinase G (PKG) and protein kinase C (PKC) inhibitors. Our data indicate that low NO bioavailability increases CD-renin synthesis and secretion, which may contribute to the activation of intrarenal renin angiotensin system.

Cholesterol induces renal vasoconstriction and anti-natriuresis by inhibiting nitric oxide production in anesthetized rats.

Although hypercholesterolemia is implicated in the pathophysiology of many renal disorders as well as hypertension, its direct actions in the kidney are not yet clearly understood. In the present study, we evaluated renal responses to administration of cholesterol (8 microg x min(-1).100 g body wt(-1); bound by polyethylene glycol) into the renal artery of anesthetized male Sprague-Dawley rats. Total renal blood flow (RBF) was measured by a Transonic flow probe, and glomerular filtration rate (GFR) was determined by Inulin clearance. In control rats (n = 8), cholesterol induced reductions of 10 +/- 2% in RBF [baseline (b) 7.6 +/- 0.3 microg x min(-1).100 g(-1)], 17 +/- 3% in urine flow (b, 10.6 +/- 0.9 microg x min(-1).100 g(-1)), 29 +/- 3% in sodium excretion (b, 0.96 +/- 0.05 mumol.min(-1).100 g(-1)) and 24 +/- 2% in nitrite/nitrate excretion (b, 0.22 +/- 0.01 nmol.min(-1).100 g(-1)) without an appreciable change in GFR (b, 0.87 +/- 0.03 ml.min(-1).100 g(-1)). These renal vasoconstrictor and anti-natriuretic responses to cholesterol were absent in rats pretreated with nitric oxide (NO) synthase inhibitor, nitro-l-arginine methylester (0.5 microg x min(-1).100 g(-1); n = 6). In rats pretreated with superoxide (O(2)(-)) scavenger tempol (50 microg x min(-1).100 g(-1); n = 6), the cholesterol-induced renal responses remained mostly unchanged, although there was a slight attenuation in anti-natriuretic response. This anti-natriuretic response to cholesterol was abolished in furosemide-pretreated rats (0.3 microg x min(-1).100 g(-1); n = 6) but remained unchanged in amiloride-pretreated rats (0.2 microg x min(-1).100 g(-1); n = 5), indicating that Na(+)/K(+)/2Cl(-) cotransport is the dominant mediator of this effect. These data demonstrate that cholesterol-induced acute renal vasoconstrictor and antinatriuretic responses are mediated by a decrease in NO production. These data also indicate that tubular effect of cholesterol on sodium reabsorption is mediated by the furosemide sensitive Na(+)/K(+)/2Cl(-) cotransporter.

Superoxide and its interaction with nitric oxide modulates renal function in prehypertensive Ren-2 transgenic rats.

OBJECTIVE: The present study was performed to examine the role of superoxide (O2*) and its interaction with nitric oxide (NO) in the regulation of renal function in prehypertensive heterozygous Ren-2 transgenic rats (TGR). METHODS: Renal responses to the O2* scavenger, tempol (150 microg/min per 100 g), and/or the NO synthase inhibitor, nitro-L-arginine methylester (L-NAME; 5 microg/min per 100 g), infused alone or in combination directly into the renal artery were evaluated in anesthetized heterozygous male TGR and aged-matched Hanover Sprague-Dawley rats (HanSD). RESULTS: There were no differences in arterial pressure (122 +/- 3 versus 115 +/- 2 mmHg), renal plasma flow (RPF; 2.09 +/- 0.1 versus 2.07 +/- 0.1 ml/min per g), glomerular filtration rate (GFR; 0.73 +/- 0.1 versus 0.74 +/- 0.1 ml/min per g) or sodium excretion (0.63 +/- 0.13 versus 0.67 +/- 0.16 micromol/min per g) between TGR and HanSD. Tempol alone caused significant increases in RPF and GFR (10 +/- 4% and 12 +/- 2%, respectively) in TGR but not in HanSD. Tempol also caused greater sodium excretory responses in TGR compared to HanSD (112 +/- 16% versus 43 +/- 7%; P < 0.05). 8-Isoprostane excretion was significantly higher in TGR than in HanSD (10.2 +/- 0.8 versus 6.5 +/- 0.7 pg/min per g), which was attenuated by tempol. L-NAME caused greater decreases in RPF and GFR in TGR (-34 +/- 4% and -22 +/- 4%, respectively) than in HanSD (-19 +/- 3% and -10 +/- 4%, respectively). Co-infusion of tempol partially attenuated the renal hemodynamic and excretory responses to L-NAME in TGR. CONCLUSIONS: These data suggest that the enhanced O2* activity and its interaction with NO during the prehypertensive phase in TGR modulates renal hemodynamic and excretory function, which may contribute to the development of hypertension in this transgenic rat model.

Evidence for Prohypertensive, Proinflammatory Effect of Interleukin-10 During Chronic High Salt Intake in the Condition of Elevated Angiotensin II Level.

IL-10 (interleukin-10) has been suggested to play a protective role in angiotensin II (AngII)-induced cardiovascular disorders. This study examined the role of endogenous IL-10 in salt-sensitive hypertension and renal injury induced by AngII. Responses to chronic AngII (400 ng/min per kilogram body weight; osmotic minipump) infusion were evaluated in IL-10 gene knockout mice fed with either normal salt diet (0.3% NaCl) or high salt (HS; 4% NaCl) diet, and these responses were compared with those in wild-type mice. Normal salt diets or HS diets were given alone for the first 2 weeks and then with AngII treatment for an additional 2 weeks (n=6 in each group). Arterial pressure was continuously monitored by implanted radio-telemetry, and a 24-hour urine collection was performed by metabolic cages on the last day of the experimental period. Basal mean arterial pressure was lower in IL-10 gene knockout mice than in wild-type (98±3 versus 113±3 mm Hg) mice. Mean arterial pressure responses to normal salt/HS alone or to the AngII+normal salt treatment were similar in both strains. However, the increase in mean arterial pressure induced by the AngII+HS treatment was significantly lower in IL-10 gene knockout mice (15±5% versus 37±3%) compared with wild-type mice. Renal tissue endothelial nitric oxide synthase expression (≈3-folds) and urinary excretion of nitric oxide metabolites, nitrate/nitrite (1.2±0.1 versus 0.2±0.02 µmol/L/24 hours) were higher in IL-10 gene knockout mice compared with wild-type mice. These results indicate that an increase in nitric oxide production helps to mitigate salt-sensitive hypertension induced by AngII and suggest that a compensatory interaction between IL-10 and nitric oxide exists in modulating AngII-induced responses during HS intake.

2-Methoxyestradiol Reduces Angiotensin II-Induced Hypertension and Renal Dysfunction in Ovariectomized Female and Intact Male Mice.

Cytochrome P450 1B1 protects against angiotensin II (Ang II)-induced hypertension and associated cardiovascular changes in female mice, most likely via production of 2-methoxyestradiol. This study was conducted to determine whether 2-methoxyestradiol ameliorates Ang II-induced hypertension, renal dysfunction, and end-organ damage in intact Cyp1b1-/-, ovariectomized female, and Cyp1b1+/+ male mice. Ang II or vehicle was infused for 2 weeks and administered concurrently with 2-methoxyestradiol. Mice were placed in metabolic cages on day 12 of Ang II infusion for urine collection for 24 hours. 2-Methoxyestradiol reduced Ang II-induced increases in systolic blood pressure, water consumption, urine output, and proteinuria in intact female Cyp1b1-/- and ovariectomized mice. 2-Methoxyestradiol also reduced Ang II-induced increase in blood pressure, water intake, urine output, and proteinuria in Cyp1b1+/+ male mice. Treatment with 2-methoxyestradiol attenuated Ang II-induced end-organ damage in intact Cyp1b1-/- and ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice and Cyp1b1+/+ male mice. 2-Methoxyestradiol mitigated Ang II-induced increase in urinary excretion of angiotensinogen in intact Cyp1b1-/- and ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice but not in Cyp1b1+/+ male mice. The G protein-coupled estrogen receptor 1 antagonist G-15 failed to alter Ang II-induced increases in blood pressure and renal function in Cyp1b1+/+ female mice. These data suggest that 2-methoxyestradiol reduces Ang II-induced hypertension and associated end-organ damage in intact Cyp1b1-/-, ovariectomized Cyp1b1+/+ and Cyp1b1-/- female mice, and Cyp1b1+/+ male mice independent of G protein-coupled estrogen receptor 1. Therefore, 2-methoxyestradiol could serve as a therapeutic agent for treating hypertension and associated pathogenesis in postmenopausal females, and in males.

Oxidant stress and blood pressure responses to angiotensin II administration in rats fed varying salt diets.

To examine the hypothesis that NAD(P)H oxidase (Nox)-derived superoxide generation is involved in the development of angiotensin II (ANG II)-induced hypertension, we evaluated the responses to ANG II infusion (65 ng/min; osmotic mini-pump) for 2 weeks in rats treated with or without apocynin (APO) (inhibitor of Nox subunits assembly) in drinking water (12 mmol/L). Rats were grouped according to their diets with varying salt content (normal salt [NS], 0.4%; high salt [HS], 8%; low salt [LS], 0.03%) given during the 2-week experimental period. The variation in salt intake did not alter mean arterial pressure (MAP, recorded via pre-implanted arterial catheter) but showed proportionate levels in urinary excretion rate of Isoprostaglandin(2alpha) (U(ISO)V; NS, 179 +/- 26; HS, 294 +/- 38; LS, 125 +/- 7 ng/kg/24 h). Treatment with ANG II increased MAP proportional to salt intake (NS, 126 +/- 3 to 160 +/- 5; HS, 116 +/- 4 to 184 +/- 5; LS, 125 +/- 1 to 154 +/- 5 mm Hg). However, ANG II increased U(ISO)V only in NS rats (250 +/- 19 ng/kg/24 h) but not in HS or LS rats. In response to ANG II, Nox subunits protein expression increased in HS but not in the NS or LS rats. Apocynin treatment partially ameliorated these changes in Nox proteins in HS rats but did not alter ANG II-induced increases in MAP or U(ISO)V. These data suggest that Nox activation may not be the sole factor or alternatively, that a constitutively active isoform of Nox is involved in oxidative stress mechanism that is associated with dietary salt or ANG II-induced hypertension.

Superoxide formation and interaction with nitric oxide modulate systemic arterial pressure and renal function in salt-depleted dogs.

To determine the role of superoxide (O(2)(-)) formation in the kidney during alterations in the renin-angiotensin system, we evaluated responses to the intra-arterial infusion of an O(2)(-) - scavenging agent, tempol, in the denervated kidney of anesthetized salt-depleted (SD, n=6) dogs and salt-replete (SR, n=6) dogs. As expected, basal plasma renin activity was higher in SD than in SR dogs (8.4 +/- 1.0 vs. 2.3 +/- 0.6 ng angiotensin 1/ml/hr). Interestingly, the basal level of urinary F(2)-isoprostanes excretion (marker for endogenous O(2)(-) activity) relative to creatinine (Cr) excretion was also significantly higher in SD compared to SR dogs (9.1 +/- 2.8 vs. 1.6 +/- 0.4 ng F(2)-isoprostanes/mg of Cr). There was a significant increase in renal blood flow (4.3 +/- 0.5 to 4.9 +/- 0.6 ml/min/g) and decreases in renal vascular resistance (38.2 +/- 5.8 to 33.2 +/- 4.7 mm Hg/ml/min/g) and mean systemic arterial pressure (148 +/- 6 to 112 +/- 10 mm Hg) in SD dogs but not in SR dogs during infusion of tempol at 1 mg/kg/min for 30 mins. Glomerular filtration rate and urinary sodium excretion (U(Na)V) did not change significantly during tempol infusion in both groups of dogs. Administration of the nitric oxide synthase inhibitor nitro-L-arginine (50 mug/kg/min) during tempol infusion caused a reduction in U(Na)V in SR dogs (47% +/- 12%) but did not cause a decrease in SD dogs. These data show that low salt intake enhances O(2)(-) activity that influences renal and systemic hemodynamics and thus may contribute to the regulation of arterial pressure in the salt-restricted state.