Alexander Castillo

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 (Gomoris 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.

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.

Decrease in IL-10 and increase in TNF-α levels in renal tissues during systemic inhibition of nitric oxide in anesthetized mice

Earlier, we demonstrated that the inhibition of nitric oxide synthase (NOS) by nitro‐l‐arginine methyl ester (l‐NAME) infusion increases the endogenous production of proinflammatory cytokine, tumor necrosis factor (TNF‐α). In the present study, we examined the hypothesis that inhibition of nitric oxide (NO) production leads to the suppression of interleukin (IL)‐10 (anti‐inflammatory cytokine) generation which facilitates the enhancement of TNF‐α production endogenously. Using appropriate enzyme‐linked immunosorbent assay kits and immunohistochemical staining, the levels of IL‐10 and TNF‐α in plasma (P) and in renal tissues (R) were measured in anesthetized mice (C57BL/6; ~10 weeks age; n = 6/group) infused with or without l‐NAME (200 μg/min/kg; i.v. for 2 h). Compared to vehicle‐treated control mice, l‐NAME‐treated mice had a lower level of IL‐10 (P, 0.3 ± 0.1 vs. 2.6 ± 0.6 ng/mL; R, 0.5 ± 0.1 vs. 3 ± 0.1 ng/mg protein) and a higher level of TNF‐α (P, 432 ± 82 vs. undetected pg/mL; R, 58 ± 7 vs. 6 ± 5 pg/mg protein). IL‐10 protein expression, present mostly in the distal nephron segments in control mice, was markedly downregulated in l‐NAME‐treated mice. Compared to control mice, TNF‐α expression increased 2.5‐fold in renal cortical sections (mostly in the distal nephron segments) in l‐NAME‐treated mice. Coinfusion of a NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP; 25 μg/min/kg) with l‐NAME in a separate group of mice prevented these changes in IL‐10 and TNF‐α induced by l‐NAME. IL‐10 infusion (0.075 ng/min/g) in l‐NAME‐treated mice markedly attenuated l‐NAME‐induced increments in TNF‐α. Thus, these results demonstrate that NOS inhibition decreases endogenous IL‐10 generation and thus, minimizes its immune downregulating action on the TNF‐α production in the kidney.

Attenuation of renal excretory responses to ANG II during inhibition of superoxide dismutase in anesthetized rats

To examine the functional interaction between superoxide dismutase (SOD) and NADPH oxidase activity, we assessed renal responses to acute intra-arterial infusion of ANG II (0.5 ng x kg(-1) x min(-1)) before and during administration of a SOD inhibitor, diethyldithiocarbamate (DETC, 0.5 mg x kg(-1) x min(-1)), in enalaprilat-pretreated (33 microg x kg(-1) x min(-1)) rats (n = 11). Total (RBF) and regional (cortical, CBF; medullary; MBF) renal blood flows were determined by Transonic and laser-Doppler flowmetry, respectively. Renal cortical and medullary tissue NADPH oxidase activity in vitro was determined using the lucigenin-chemiluminescence method. DETC treatment alone resulted in decreases in RBF, CBF, MBF, glomerular filtration rate (GFR), urine flow (V), and sodium excretion (U(Na)V) as reported previously. Before DETC, ANG II infusion decreased RBF (-18 +/- 3%), CBF (-16 +/- 3%), MBF [-5 +/- 6%; P = not significant (NS)], GFR (-31 +/- 4%), V (-34 +/- 2%), and U(Na)V (-53 +/- 3%). During DETC infusion, ANG II also caused similar reductions in RBF (-20 +/- 4%), CBF (-19 +/- 3%), MBF (-2 +/- 2; P = NS), and in GFR (-22 +/- 7%), whereas renal excretory responses (V; -12 +/- 2%; U(Na)V; -24 +/- 4%) were significantly attenuated compared with those before DETC. In in vitro experiments, ANG II (100 muM) enhanced NADPH oxidase activity both in cortical [13,194 +/- 1,651 vs. 20,914 +/- 2,769 relative light units (RLU)/mg protein] and in medullary (21,296 +/- 2,244 vs. 30,597 +/- 4,250 RLU/mg protein) tissue. Application of DETC (1 mM) reduced the basal levels and prevented ANG II-induced increases in NADPH oxidase activity in both tissues. These results demonstrate that renal excretory responses to acute ANG II administration are attenuated during SOD inhibition, which seems related to a downregulation of NADPH oxidase in the deficient condition of SOD activity.

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.

705 Suppression of plasma and renal tissue levels of interleukin-10 during infusion of a nitric oxide synthase inhibitor in anesthetized mice

Objectives: We reported earlier that increases in plasma and renal tissue levels of tumor necrosis factor-alpha (TNF&agr;) during systemic inhibition of nitric oxide synthase (NOS) modulates renal function in anesthetized mice. In the present study, we examined the hypothesis that this enhancement of TNF&agr; production is directly linked to the suppression of interleukin-10 (IL-10; an anti-inflammatory cytokine) production in response to NOS inhibition. Methods: Using appropriate ELISA kits (eBioscience, Inc.; San Diego, CA), levels of IL-10 and TNF&agr; in plasma as well as in renal tissue were measured in groups of anesthetized mice treated with or without intravenous infusion of a NOS inhibitor, nitro-L-arginine methyl ester (L-NAME; 200 &mgr;g/kg/min). IL-10 protein expression was also measured in renal tissue section by immuno-histochemical staining using IL-10 antibody (R&D Systems, Inc.; Minneapolis, MN) in these mice. Results: L-NAME infusion for more than 85 min resulted in decreases in IL-10 levels in plasma (0.7 ± 0.1 vs 2.6 ± 0.6 ng/mL; n = 6) and in renal tissue (0.8 ± 0.1 vs 1.3 ± 0.1 ng/mg protein; n = 7) compared to non-treated control mice which was associated with significant down regulation of IL-10 protein expression in renal tissues (mostly in the distal tubules). Replacement of IL-10 by infusing it (0.075 ng/g/min for 85 min) in L-NAME treated mice markedly attenuated the plasma level of TNF&agr; (53 ± 16 pg/mL; n = 7) compared to the level in mice treated with L-NAME alone (392 ± 80 pg/mL; n = 7). Conclusions: The results demonstrate that NOS inhibition decreases IL-10 production and thus, minimizes its immune downregulating action on the production of pro-inflammatory cytokines, particularly on TNF&agr;.

RENAL RESPONSES TO ANGIOTENSIN II IN MICE LACKING THE GENE FOR TUMOR NECROSIS FACTOR-α.: 354

Recent studies have implicated a role for a proinflammatory cytokine, tumor necrosis factor-α (TNF-α), in angiotensin II (ANGII)-induced renal injury and the development of salt-sensitive hypertension. To examine this relationship between TNF-α and ANGII actions in the kidney, we assessed renal responses to acute ANG II (1 ng/min/g for 30 minutes, IV) in anesthetized knockout (KO) mice lacking TNF-α and compared these responses with those of wild-type (WT) mice. Systemic blood pressure (BP) was recorded from a cannula placed in the left carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances, respectively. Compared with WT (n = 6), KO (n = 8) mice did not have significant differences in the basal values of BP (92 ± 4 and 99 ± 2 mm Hg), RBF (3.8 ± 0.3 and 3.4 ± 0.2 mL.min−1.g−1), GFR (0.74 ± 0.04 and 0.75 ± 0.03 mL.min−1.g−1), urine flow (V, 8.8 ± 0.4 and 9.9 ± 0.6 μL.min−1.g−1), and sodium excretion (UNaV, 0.87 ± 0.07 and 0.93 ± 0.12 μmol.min−1.g−1). However, KO exhibit lower urinary nitrite/nitrate (UNOxV; 0.13 ± 0.02 vs 0.35 ± 0.07 nmol.min−1.g−1) and 8-isoprostane excretion (UISOV; 1.26 ± 0.05 vs 1.97 ± 0.20 pg.min−1.g−1) compared with WT. ANGII caused similar increment in BP in both KO and WT (Δ24 ± 3 and Δ26 ± 2 mm Hg). Interestingly, ANGII caused minimal decrease in RBF (−3 ± 2%) in KO compared with WT (−30 ± 5%). Moreover, ANGII increased GFR in KO (7 [138} 2%) but not in WT. Although V and UNaV responses to ANGII were not much different between the the strains, there were lower UNOxV (92 ± 19% vs 143 [138} 52%; p

ENHANCED SUPEROXIDE ACTIVITY INDUCES SALT SENTSITIVITY IN ENDOGENOUS NITRIC OXIDE SYNTHASE KNOCKOUT MICE.: 267

Nitric oxide (NO) synthase inhibition enhances superoxide (O2−) activity. The present study was conducted to examine the hypothesis that salt sensitivity is induced by superoxide (O2−) activity that is enhanced due to a deficiency in endogenous nitric oxide (NO) production. Male knockout (KO) mice lacking the gene for endothelial NO synthase and their genetic background wild-type C57BL6J mice (WT) were fed a diet containing either normal salt (NS, 0.4% NaCl) or high salt (HS, 4% NaCl) and treated with or without a O2− scavenger, tempol (400 mg/L), in drinking water for 2 weeks. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and urine collection was performed in metabolic cages on every third day during the 2-week experimental period. Urinary 8-isoprostane concentration (a marker for oxidative stress) was measured by ELISA kit (Cayman). Baseline SBP values in KO groups were significantly higher compared with WT groups (138 ± 5 vs 116 ± 4 mm Hg). At the end of the 2-week period, there was no significant difference in SBP between the tempol-treated and untreated WT groups with NS intake. However, SBP was significantly lower in tempol-treated KO mice compared with untreated KO mice fed on NS diets (131 ± 2 vs 142 ± 3 mm Hg). HS intake had no effect on SBP in untreated and treated WT groups; however, SBP was significantly increased in KO with HS intake (154 ± 4 mm Hg) over the 2-week period. Tempol significantly attenuated the effect of HS on SBP in the KO group (139 ± 2 mm Hg). There were no differences in baseline urinary 8-isoprostane excretion (UISOV) between WT and KO mice. However, at the end of 2 weeks, compared with WT and KO with NS intake (2.2 ± 0.1 and 2.4 ± 0.3 ng/d, respectively), UISOV was increased in both WT and KO groups with HS intake (2.9 ± 0.3 and 3.6 ± 0.2 ng/d, respectively), the increase in UISOV being greater in KO compared with WT. Tempol attenuated UISOV in both WT and KO with HS intake (2.5 ± 0.1 and 2.9 ± 0.2 ng/d). These data suggest that, in the condition of eNOS enzyme deficiency, dietary high-salt intake induces enhanced O2− activity that contributes to the development of salt-sensitive hypertension.

432 RENAL AND SYSTEMIC RESPONSES TO NITRIC OXIDE BLOCKADE IN GP91PHOX KNOCKOUT MICE

An interactive role of nitric oxide (NO) and superoxide (O2-) in the regulation of renal function has been suggested in recent studies. NAD(P)H oxidase is known to be a major source of O2- production in the kidney. To understand further this NO-O2- interaction in the kidney, we have assessed the renal responses to systemic administration of NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME; 0.2 μg/min/g BW) in inactin anesthetized knockout (KO, n = 8) mice, deficient of gene for gp91phox and compared these responses to that in wild-type (WT, C57BL/6, n = 7) mice. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances respectively. Baseline mean arterial blood pressure (BP) recorded from a carotid arterial cannula was not different between KO (91 ± 4 mm Hg) and WT mice (92 ± 6 mm Hg). Basal RBF was higher in KO compared to WT (5.4 ± 0.5 vs. 4.3 ± 0.3 mL.min-1.g-1, p < .05). However, basal levels of renal vascular resistance (RVR, 18.5 ± 2.5 and 22.2 ± 1.8 mm Hg.mL-1.min-1.g-1), GFR (0.56 ± 0.06 and 0.64 ± 0.06 mL.min-1.g-1), urine flow (V, 6.9 ± 1.5 and 9.5 ± 2.2 μL.min-1.g-1) and sodium excretion (UNaV, 0.67 ± 0.1 and 0.84 ± 0.3 μmoL.min-1.g-1) were not significantly different between KO and WT mice respectively. In WT mice, L-NAME infusion for 60 minutes resulted in a reduction of 17 ± 4 % in RBF and an increase of 55 ± 9% in RVR. Interestingly, L-NAME caused less reduction in RBF (10 ± 3%) and less increases in RVR 34 ± 6% in KO mice compared to WT. There were no changes in GFR during L-NAME infusion in both the KO (0.56 ± 0.06 mL.min-1.g-1) and in WT (0.69 ± 0.08 mL.min-1.g-1) mice. Systemic infusion of L-NAME also caused increases in BP (109 ± 5 and 115 ± 4 mm Hg), V (21 ± 4 and 30 ± 5 μL.min-1.g-1) and UNaV (4.0 ± 0.8 and 5.5 ± 1.0 μmoL.min-1.g-1) in KO and WT mice respectively. Although the excretory responses to L-NAME were generally lower in KO mice compared to WT mice, but these changes were not significantly different. L-NAME increased urinary excretion of 8-isoprostane in WT (4.7 ± 0.7 to 8.7 ± 1.2 pg/min/g, p < .05) but not in KO mice (4.8 ± 0.6 to 5 ± 0.4 pg/min/g) indicating increased O2- activity in WT mice during NO blockade. These data further support the notion that renal responses to NO blockade are influenced by enhancement of O2- activity induced by NAD(P)H oxidase.