Expression Of NADPH Oxidase Components Research Articles

A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

The Serine Protease Inhibitor Camostat Mesilate Attenuates the Progression of Chronic Kidney Disease through its Antioxidant Effects

Background/Aims: We have so far demonstrated the renoprotective effect of camostat mesilate (CM) in 5/6 nephrectomized rats at least partly through its antioxidant effect. However, precise mechanisms were not fully clarified. Therefore, we now examined the renoprotective and antioxidant mechanisms of CM by using the adenine-induced chronic kidney disease (CKD) rat model. Methods: In protocol 1, we analyzed the effect of CM on CKD. Rats were fed on a 0.75% adenine diet for 3 weeks to induce CKD followed by the experimental period with vehicle, CM, or hydralazine (HYD) treatment for 5 weeks. In protocol 2, we examined the safety of CM and HYD on the normal rats. In addition, we explored free radical scavenging activities of CM and its metabolites in vitro using electron paramagnetic resonance (EPR) spectroscopy. Results: CM, but not HYD, significantly reduced the serum creatinine levels, although both treatments showed similar reduction in the blood pressure. CM decreased mRNA expression and protein levels of fibrotic markers, the severity of renal fibrosis, the accumulation of oxidative stress, and the expression of NADPH oxidase components in the kidney. In the protocol 2, there were no statistically significant differences in general parameters except for the systolic blood pressure in HYD group. EPR study revealed that CM and its metabolites have potent hydroxyl radical scavenging activities in vitro. Conclusion: Our findings indicate that CM significantly ameliorates the progression of CKD partly through its antioxidant effect independently from its blood pressure-lowering effect. Our results suggest the possibility that CM could be a new therapeutic agent that could arrest the progression of CKD.

Calcium Channel Blocker Enhances Beneficial Effects of an Angiotensin II AT1 Receptor Blocker against Cerebrovascular-Renal Injury in type 2 Diabetic Mice

Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-Ay mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-Ay mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.

Abstract 8857: Iron Restriction Prevents the Progression of Diabetic Nephropathy in db/db Mice

Background: Iron is well-known as an essential trace metal for all living body. On the other hand, excess iron causes oxidative stress through catalyzing highly toxic hydroxyl radical via Fenton/Haber-Weiss reaction. Recent studies have shown that iron reduction ameliorates diabetic condition and prevents development of adipocyte hypertrophy. In the present study, we studied the protective effect of iron reduction against diabetic nephropathy, a diabetic complication. Methods and Results: We employed male db/db mice, a model of diabetic nephropathy and male db/m mice as control mice. db/db mice were divided into 2 groups, normal diet group (ND)(Fe 100mg/kg food) and iron-restricted diet group (ID)(Fe 10mg/kg food). Urinary albumin excretion was increasing in db/db mice with ND compared with db/m mice with ND, which was suppressed in db/db mice with ID. In renal histological analysis, increased glomerular volume and mesangial area expansion in ND-fed db/db mice were reduced in ID-fed db/db mice. Deposition of extracelluar matrix such as collagen IV and fibronectin was decreased in db/db mice with ID. Upregulated glomerular desmin expression in db/db mice with ND was diminished in db/db mice with ID. In terms with oxidative stress, ID diminished increased superoxide production in kidney of ND-fed db/db mice. Renal expression of NADPH oxidase components, p22phox and NOX4, were augmented in db/db mice with ND-fed, which was abolished by ID-fed. In terms with renal iron transporter, there were no differences of expression of transferrin receptor and divalent metal transporter-1 between db/m mice and db/db mice, increased ferroportin expression in db/db mice with ND- was reversed by ID to the same level as db/m mice. Urinary iron excretion was significantly higher in ND-fed db/db mice, which was reduced with ID administration. Conclusion: These findings suggest that dietary iron restriction exert preventive effect against development of diabetic nephropathy via the reduction of oxidative stress.

Expression of NADPH oxidase in human pancreatic islets

AimsNADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human β-cell function were investigated. Main methods and key findingsIn isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22PHOX, p47PHOX and p67PHOX), Western blotting (p47PHOX and p67PHOX) and immunohistochemistry (p47PHOX, p67PHOX and gp91PHOX). Immunohistochemistry experiments showed co-localization of p47PHOX, p67PHOX and gp91PHOX (isoform 2 of NADPH oxidase—NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SignificanceThese findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human β-cell function.

Angiotensin II-NADPH oxidase-derived superoxide mediates diabetes-attenuated cell excitability of aortic baroreceptor neurons

Overactivation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is involved in diabetes-depressed excitability of aortic baroreceptor neurons in nodose ganglia. This involvement links to the autonomic dysfunction associated with high morbidity and mortality in diabetic patients. The present study examined the effects of an angiotensin II type I receptor (AT(1)R) antagonist (losartan), a NADPH oxidase inhibitor (apocynin), and a superoxide dismutase mimetic (tempol) on the enhanced HCN currents and attenuated cell excitability in diabetic nodose neurons. In sham and streptozotocin-induced type 1 diabetic rats, HCN currents and cell excitability of aortic baroreceptor neurons were recorded by the whole cell patch-clamp technique. The angiotensin II level in nodose ganglia from diabetic rats was higher than that from sham rats (101.6 ± 4.8 vs. 38.9 ± 4.2 pg/mg protein, P < 0.05). Single-cell RT-PCR, Western blot, immunofluorescence staining, and chemiluminescence data showed that mRNA and protein expression of AT(1)R, protein expression of NADPH oxidase components, and superoxide production in nodose neurons were increased in diabetic rats compared with those from sham rats. HCN current density was higher and cell excitability was lower in aortic baroreceptor neurons from diabetic rats than that from sham rats. Losartan (1 μM), apocynin (100 μM), and tempol (1 mM) normalized the enhanced HCN current density and increased the cell excitability in the aortic baroreceptor neurons of diabetic rats. These findings suggest that endogenous angiotensin II-NADPH oxidase-superoxide signaling contributes to the enhanced HCN currents and the depressed cell excitation in the aortic baroreceptor neurons of diabetic rats.

Involvement of NADPH oxidase in up-regulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice

The present study demonstrated that oxidized LDL (oLDL) increased the generation of superoxide and hydrogen peroxide (H2O2), the abundances of NADPH oxidase (NOX)4, NOX2, p22-phox and lectin-like oLDL receptor-1 (LOX-1) in wild-type or heat shock factor-1 (HSF1)-deficient mouse embryo fibroblasts (MEF). LOX-1 antibody inhibited LDL or oLDL-induced expression of NOX components in MEF. Abundance of HSF1 or plasminogen activator inhibitor-1 (PAI-1) was increased by oLDL in wild-type, but not in HSF1-deficient MEF. Diphenyleneiodonium or siRNA for NOX or p22-phox inhibited oLDL-induced increases of HSF1, PAI-1 and H2O2 in MEF. Increased NOX4, NOX2, LOX1, HSF1 and PAI-1 were detected in aortae and hearts of apolipoprotein E-knockout (apoE-KO) mice compared to controls, which were associated with increased serum cholesterol or plasma PAI-1. The results suggest that NOX is required for oLDL-induced HSF1 or PAI-1 expression in MEF, which was supported by the up-regulation of NOX, LOX-1, HSF1 and PAI-1 in apoE-KO mice.

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation

Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.

High dietary protein intake induces endothelial dysfunction in uninephrectomized rats

High dietary protein (HP) intake is a risk factor for chronic kidney disease (CKD). HP intake is associated with the development of albuminuria and glomerulosclerosis in uninephrectomized rats. In such rats, we investigated whether HP intake induces endothelial dysfunction. Male Wistar rats were divided into sham-operated rats fed a standard-protein diet, sham-operated rats fed a high-protein diet, uninephrectomized rats fed a standard-protein diet (NxSP) and uninephrectomized rats fed a high-protein diet (NxHP) (n=8 each). One week after treatment, endothelial function and urinary albumin excretion (UAE) were measured. Protein expression, phosphorylation at serine residue 1177 and uncoupling of endothelial nitric oxide synthase (eNOS), and mRNA expression of NADPH oxidase components were assessed in the aorta. NxHP rats showed hypertriglyceridemia and modest hyperhomocysteinemia. Endothelial function was significantly lower, and UAE was significantly higher in NxHP rats compared with the other groups (P<0.01 each), although there was no difference in creatinine clearance between NxSP and NxHP rats. Expression levels, phosphorylation and the dimer/monomer ratio of eNOS did not differ among the four groups. HP intake did not modify p22phox and p47phox expression levels in uninephrectomized rats. In conclusion, HP intake induced endothelial dysfunction and enhanced albuminuria in uninephrectomized rats, inde-pendent of renal function, suggesting that HP intake may cause the development of cardiovascular disease associated with CKD.

Vascular biology of angiotensin and the impact of physical activity

The renin-angiotensin system (RAS) is important for regulating blood pressure and extracellular fluid. The concept of the RAS has recently evolved from a classical systemic endocrine system to an appreciation of local RASs functioning in a paracrine manner, including in the vascular wall. Angiotensin II (AII), the main effector of the RAS, is a potent vasoconstrictor formed by the action of angiotensin-converting enzyme (ACE). ACE is multifunctional and also destroys the endogenous vasodilator bradykinin. A recently discovered novel ACE2 enzyme is responsible for forming a vasodilatory compound, angiotensin 1-7, from AII. Thus, the actions of ACE and ACE2 are antagonistic. Tissue actions of AII are mediated by specific receptors, AT1 and AT2, with AT1 mediating the classical actions. AT1-stimulated vasoconstricton occurs via phospholipase-D-mediated second messenger generation directly, and indirectly via the coupling of AT1 to the prooxidant enzyme NADPH oxidase. Since the vascular NADPH oxidase is a major source of vascular reactive oxygen species generation and is responsible for the breakdown of the vasodilator nitric oxide (NO), there is another potential link between RAS and regulation of vasodilatory pathways. AT2 signaling is antagonistic to AT1 signaling, and results in bradykinin and NO formation. Chronic AII signaling induces vascular dysfunction, whereas pharmacological management of the RAS can not only control blood pressure, but also correct endothelial dysfunction in hypertensives. Exercise training can also improve endothelial function in hypertensives, raising the question of whether there is a potential role for RAS in mediating the vascular effects of exercise training. Recent studies have demonstrated reductions in the expression of NADPH oxidase components in the vascular wall in response to exercise training, thus tempering one of the main cellular effectors of AII, and this is associated with reduced vascular ROS production and enhanced NO bioavailability. Importantly, it has now been demonstrated in human arteries that exercise training also tempers vascular AT1 receptor expression and AII-induced vasoconstriction, while enhancing endothelium-dependent dilation. The signals responsible for these chronic adaptations are not clearly understood, and may include changes in RAS components prompted by acute exercise. ACE genotype may have an effect on physical activity levels and on the cardiovascular responses to exercise training, and the II genotype (compared with ID and DD) is associated with the largest endothelium-dependent dilations in athletes compared with those in sedentary individuals. Thus, the tissue location of the RAS, the complement of ACE/ACE2, the receptor expression of AT1/AT2, and the ACE genotype are all variables that could impact the vascular responses to exercise training, but the responses of most of these variables to regular exercise training and the mechanisms responsible have not been systematically studied.

Role of Aldosterone in Oxidative Stress and Renal Injury

Recent clinical studies have indicated the utility of mineralocorticoid receptor (MR) antagonists in cardiovascular and renal injuries. Chronic treatment with aldosterone/salt resulted in severe cardiac and renal injuries in rats. Further studies showed that the aldosterone-induced organ injuries were associated with increases in expression of NADPH oxidase components and reactive oxygen species (ROS) levels. Treatment with a selective MR antagonist, eplerenone, prevented the elevation of ROS levels and ameliorated organ injuries. In vitro studies also showed that MR is highly expressed in cultured vascular smooth muscle cells, glomerular mesangial cells and renal fibroblasts. In these cells, aldosterone-induced cell injuries were associated with increases in NADPH oxidase activity and superoxide generation. Further, the aldosterone-dependent cell injuries were markedly attenuated by treatment with eplerenone. These accumulating data support the notion that the aldosterone/MR is involved in the pathogenesis of cardiovascular and renal injuries through NADPH oxidase-dependent ROS production.

Up‐regulation of NADPH oxidase components and increased production of interferon‐gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P=0.036). Monocytes from SCD patients showed higher levels of p47(phox) phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P=0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P>0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Pigment Epithelium-Derived Factor Inhibits Neointimal Hyperplasia after Vascular Injury by Blocking NADPH Oxidase-Mediated Reactive Oxygen Species Generation

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22(phox) and gp91(phox), in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G(1) cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22(PHOX) and gp91(PHOX). PEDF down-regulated G(1) cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty.

Exercise training raises expression of the cytosolic components of NADPH oxidase in rat neutrophils

Exercise activates neutrophil burst and this effect is dependent on training status and exercise intensity. In this study, the chronic effect of treadmill exercise on phagocytosis, production of reactive oxygen metabolites and expression of NADPH oxidase components in rat neutrophils was investigated. Neutrophils were obtained by intraperitoneal lavage with PBS. After 11 weeks of training the exercised group showed increased phagocytosis capacity (49%) and production of reactive oxygen metabolites (6.6-fold) when compared with neutrophils from the sedentary group. Exercised had no effect on expression of the membrane components of NADPH oxidase (p22( phox ), gp91( phox )). In contrast, there was an increase of the p47( phox ) mRNA levels (by 126%), the cytosolic component of the enzyme. In addition, exercise increased the protein content of p47( phox ) (by 22%) and of p67( phox ) (by 2.8-fold) in neutrophils. Evidence is then presented that training to moderate exercise increases phagocytosis and production of reactive oxygen metabolites and the expression of p47( phox ) and p67( phox ) in neutrophils. Therefore, moderate exercise might enable neutrophils to respond more efficiently when exposed to pathogens.

Oral Administration of Tetrahydrobiopterin Slows the Progression of Atherosclerosis in Apolipoprotein E-Knockout Mice

Although it has been reported that oral administration of tetrahydrobiopterin (BH4) prevents endothelial dysfunction and vascular oxidative stress in various rat models, the effect of treatment with BH4 on atherogenesis remains unclear. In this study, we investigated whether oral BH4 treatment might slow the progression of atherosclerosis using hypercholesterolemic apolipoprotein E-knockout mice. We report that ingesting BH4 in drinking water is sufficient to inhibit atherogenesis in mice. Furthermore, we report that BH4 treatment improves endothelial dysfunction and attenuates increased mRNA expression of NADPH oxidase components, as well as a number of inflammatory factors, such as LOX-1 and MCP-1, in the aortas of apolipoprotein E- knockout mice. Strategies such as oral administration of BH4 to ensure continuous BH4 availability may be effective in restoring nitric oxide-mediated endothelial function and limiting vascular disease and the progression of atherosclerosis.

Tetrahydrobiopterin Slows the Progression of Atherosclerosis

Abstract We investigated whether oral tetrahydrobiopterin (BH4) treatment might slow the progression of atherosclerosis using hypercholesterolemic ApoE-knockout (KO) mice. We report that ingesting BH4 in drinking water is effective to inhibit atherogenesis in mice. Furthermore, we report that BH4 treatment improves endothelial dysfunction and attenuates increased mRNA expression of NADPH oxidase components, as well as a number of inflammatory factors, such as LOX-1 and MCP-1, in the aortas of ApoE-KO mice. Strategies such as oral administration of BH4 to ensure continuous BH4 availability may be effective in restoring NO-mediated endothelial function and limiting vascular disease and the progression of atherosclerosis.

Exercise training raises expression of the cytosolic components of NADPH oxidase in rat neutrophils

Exercise activates neutrophil burst and this effect is dependent on training status and exercise intensity. In this study, the chronic effect of treadmill exercise on phagocytosis, production of reactive oxygen metabolites and expression of NADPH-oxidase components in rat neutrophils was investigated. Neutrophils were obtained by intraperitoneal lavage with PBS. After 11 weeks of training the exercised group showed increased phagocytosis capacity (49%) and production of reactive oxygen metabolites (6.6 fold) when compared with neutrophils from the sedentary group. Exercised had no effect on expression of the membrane components of NADPH-oxidase (p22phox, gp91phox). In contrast, there was an increase of the p47phox mRNA levels (by 126%), the cytosolic component of the enzyme. In addition, exercise increased the protein content of p47phox (by 22%) and of p67phox (by 2.8 fold) in neutrophils. Evidence is then presented that training to moderate exercise increases phagocytosis and production of reactive oxygen metabolites and the expression of p47phox and p67phox in neutrophils. Therefore, moderate exercise might enable neutrophils to respond more efficiently when exposed to pathogens. Financial support: FAPESP, CNPq and CAPES.

Interferon-γ enhances superoxide production in human mesangial cells via the JAK–STAT pathway

Immune reactive cytokines, such as interferon (IFN)-gamma, have multiple effects in glomerulonephritis. Superoxide anions (O(2)(-)), which are associated with the progression of glomerulonephritis, are mainly generated by nicotinamide adenine dinucleotide phosphate (reduced form) NAD(P)H oxidases. We determined the effects of IFN-gamma on O(2)(-) production, phosphorylation of signal transducer and activator of transcription (STAT)-1alpha, and the mRNA and protein expressions of p22phox and Nox1, components of NAD(P)H oxidases, in human mesangial cells (HMCs). Significant increases in O(2)(-) production with IFN-gamma were completely abolished by the flavin-containing enzyme inhibitor, diphenyleneiodonium (10 micromol/l), and the Janus-activated kinase (JAK)2 inhibitor, AG490 (100 micromol/l). Phosphorylated STAT-1alpha was detected after 5 min of IFN-gamma stimulation using Western blot analysis, and binding to the gamma-activating site was observed from 30 min to 4 h, thereafter by electrophoretic mobility shift assay (EMSA). Super-shift analysis in EMSA revealed that the main transcription factor was STAT-1alpha. IFN-gamma significantly increased the expression of p22phox mRNA and protein, although expression was inhibited by AG490. These data suggest that IFN-gamma stimulates O(2)(-) production in HMCs via the JAK-STAT pathway and NAD(P)H oxidase.

Addition of the Antioxidant Probucol to Angiotensin II Type I Receptor Antagonist Arrests Progressive Mesangioproliferative Glomerulonephritis in the Rat

Angiotensin II (Ang II) and reactive oxidative species (ROS) that are produced by NADPH oxidase have been implicated in the progression of glomerulonephritis (GN). This study examined the effect of simultaneously interrupting Ang II and ROS with an Ang II receptor blocker (ARB), candesartan, and a free radical scavenger, probucol, in a model of progressive mesangioproliferative GN induced by the injection of anti-Thy-1 antibody into uninephrectomized rats. Nephritic rats were divided into four groups and given daily oral doses of the following: Vehicle, 1% probucol diet, 70 mg/L candesartan in drinking water, and probucol plus candesartan. These treatments lasted until day 56. Vehicle-treated nephritic rats developed progressively elevated proteinuria and glomerulosclerosis. Candesartan kept proteinuria significantly lower than vehicle or probucol. The addition of probucol to candesartan normalized urinary protein excretion. Increases in BP in nephritic rats were lowered by these treatments, except with probucol. It is interesting that both glomerular cell number and glomerulosclerosis were significantly decreased by candesartan and normalized by the addition of probucol. Immunohistochemical studies for TGF-beta1, collagen type I, and fibronectin revealed that the combined treatment abolished glomerular fibrotic findings compared with candesartan. In addition, glomerular expression of NADPH oxidase components and superoxide production suggested that the combined treatment completely eliminated NADPH oxidase-associated ROS production. In conclusion, our study provides the first evidence that the antioxidant probucol, when added to an Ang II receptor blockade, fully arrests proteinuria and disease progression in GN. Furthermore, the data suggest that NADPH oxidase-associated ROS production may play a pivotal role in the progression of GN. The combination of probucol and candesartan may represent a novel route of therapy for patients with progressive GN.

Resistance to oxidative stress by chronic infusion of angiotensin II in mouse kidney is not mediated by the AT2receptor

Wild-type mice are resistant to ANG II-induced renal injury and hence form an attractive model to study renal defense against ANG II. The present study tested whether ANG II induces expression of antioxidative genes via the AT2 receptor in renal cortex and thereby counteracts prooxidative forces. ANG II was infused in female C57BL/6J mice for 28 days and a subgroup received AT2 receptor antagonist (PD-123,319) for the last 3 days. ANG II induced hypertension and aortic hypertrophy; proteinuria and renal injury were absent. Urinary nitric oxide metabolites (NOx) were decreased, and lipid peroxide (TBARS) excretion remained unchanged. Expression of NADPH oxidase components was decreased in renal cortex but induced in aorta. Heme oxygenase-1 (HO-1) was induced in both renal cortex and aorta. In contrast, ANG II suggestively increased AT2 receptor expression in kidney but not in aorta. AT2 receptor blockade enhanced hypertension in ANG II-infused mice, reversed ANG II effects on NOx excretion, but did not affect TBARS. Despite its prohypertensive effect, expression of prooxidative genes in the renal cortex decreased rather than increased after short-term AT2 receptor blockade and renal HO-1 induction after ANG II was normalized. Thus chronic ANG II infusion in mice induces hypertension but not oxidative stress. In contrast to the response in aorta, gene expression of components of NADPH-oxidase was not enhanced in renal cortex. Although ANG II administration induced renal cortical AT2 receptor expression, blockade of that receptor did not unveil the AT2 receptor as intrarenal dampening factor of prooxidative forces.