Nox4 NAD(P)H Oxidase Mediates Hypertrophy and Fibronectin Expression in the Diabetic Kidney*

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy (DN) 4 (1)(2)(3)(4). Whole kidneys, glomer-uli, and tubules undergo hypertrophy by increase in cell size and accumulation of extracellular matrix (3,4). Hypertrophy of the glomerular and tubular compartments precedes the development of irreversible renal changes in diabetes including glomerulosclerosis and tubulointerstitial fibrosis (3,5). Data from animal models as well as cultured renal cells indicate that hyperglycemia and high glucose induce hypertrophy and extracellular matrix expansion (3,6,7).
Oxidative stress has emerged as a critical pathogenic factor in the development of DN (8 -11). Diabetes is accompanied by increased generation of reactive oxygen species (ROS) in tissues including the kidney (12)(13)(14)(15). However, the results of treatment with antioxidants have been inconclusive (16). Although multiple pathways may result in ROS generation, recent studies indicate that a multicomponent phagocyte-like NAD(P)H oxidase is a major source of ROS in many nonphagocytic cells, including renal cells such as tubular epithelial cells and glomerular mesangial cells (MCs) (17)(18)(19). Under physiologic conditions, NAD(P)H oxidases have a very low constitutive activity that can be up-regulated in response to various stimuli (15, 17, 20 -22). For instance, it has been reported that enhanced NAD(P)H oxidase activity is associated with oxidative damage to DNA in diabetic glomeruli (23,24). These NAD(P)H oxidases are isoforms of the neutrophil oxidase, in which the catalytic subunits, termed Nox proteins, correspond to homologues of gp91 phox (or Nox2), the catalytic moiety found in phagocytes (17,20). In this family, Nox4, which appears to share the same overall structure with gp91 phox /Nox2, is abundant in the vascular system, kidney cortex, and MCs (17,20,(25)(26)(27). However, the biological role(s) of Nox4 is not well understood at present. It has been proposed that Nox4, a major source of ROS in the vasculature and the kidney, could function under pathologic conditions (20,22,24). We have reported previously that Nox4-derived ROS mediate angiotensin II (Ang II)-induced signaling and protein synthesis in mesangial cells (27,28), suggesting its potential involvement in kidney hypertrophy under pathologic conditions.
In this study, we determined whether Nox4 mediates ROS generation induced by diabetes in vivo and by high glucose in cultured cells. Antisense oligonucleotides for Nox4 were administered to a rat model of streptozotocin-induced type 1 diabetes and to cultured cells in vitro, and their effects on oxidative stress, Akt/protein kinase B (PKB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, renal hypertrophy, and fibronectin expression were investigated.

Animals and Treatments
Male Sprague-Dawley rats weighing between 200 and 225 g were divided into four groups of 10 rats/group. Group 2 was injected intravenously via the tail vein with 55 mg/kg body weight streptozotocin (STZ) in sodium citrate buffer (0.01 M, pH 4.5) to induce diabetes. Group 1 was injected with an equivalent amount of sodium citrate buffer alone. Rats in groups 3 and 4 were injected with STZ followed by either phosphorothioated sense or antisense (AS) oligonucleotides for Nox4 (90 ng/g body weight/day) administered subcutaneously by an ALZET osmotic pump for 14 days (ALZA, Palo Alto, CA). Oligonucleotides were administered 72 h after STZ injection for 14 days. Blood glucose concentration (LifeScan One Touch glucometer (Johnson & Johnson)) was monitored 24 h later and periodically thereafter. Three additional groups of control, diabetic, and diabetic rats treated with insulin were also studied. Twenty-four h after STZ injection, diabetic rats were treated daily with 4 -6 units of NPH insulin supplemented with regular insulin (Novo Nordisk Pharmaceuticals Inc., Princeton, NJ) subcutaneously. All rats had unrestricted access to food and water and were maintained in accordance with Institutional Animal Care and Use Committee procedures.
At day 14, all rats were euthanized, and both kidneys were removed and weighed. A slice of kidney cortex at the pole was embedded in paraffin or flash-frozen in liquid nitrogen for light microscopy and image analyses. In addition, cortical tissue was used for isolation of glomeruli by differential sieving as described (29), and samples of cortical tissue were frozen for biochemical analyses. NAD(P)H oxidase activity measurements were performed on freshly obtained tissue.
Antisense oligonucleotides were designed near the ATG start codon of rat Nox4 (5Ј-AGCTCCTCCAGGACAGCGCC-3Ј) (27,28). Antisense and the corresponding sense oligonucleotides were synthesized as phosphorothioated oligonucleotides and purified by high performance liquid chromatography (Advanced Nucleic Acid Core Facility, University of Texas Health Science Center at San Antonio).

Cell Culture and Transfections
Rat glomerular MCs were isolated and characterized as described (30). These cells were used between the 15th and 30th passages. Selected experiments were performed in primary and early passaged MCs to confirm the data obtained with late passages. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with antibiotic/antifungal solution and 17% fetal bovine serum. Transient transfection of antisense and sense oligonucleotides for Nox4 was performed by electroporation or with Lipofectamine as described previously (27,28).

NADPH Oxidase Assay
NADPH oxidase activity was measured by the lucigenin-enhanced chemiluminescence method.
Kidney Cortex and Glomeruli-Homogenates from renal cortex or isolated glomeruli were prepared in 1 ml and 500 l, respectively, of lysis buffer (20 mM KH 2 PO 4 , pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 0.5 g/ml leupeptin) by using a Dounce homogenizer (100 strokes on ice). Homogenates were subjected to a low speed centrifugation at 800 ϫ g, 4°C, for 10 min to remove the unbroken cells and debris, and aliquots were used immediately. To start the assay, 100 l of homogenates were added to 900 l of 50 mM phosphate buffer, pH 7.0, containing 1 mM EGTA, 150 mM sucrose, 5 M lucigenin, and 100 M NADPH. Photon emission in terms of relative light units was measured every 20 or 30 s for 10 min in a luminometer. There was no measurable activity in the absence of NADPH. A buffer blank (less than 5% of the cell signal) was subtracted from each reading. Superoxide production was expressed as relative chemiluminescence (light) units (RLU)/mg protein. Protein content was measured using the Bio-Rad protein assay reagent.
Cultured Mesangial Cells-NADPH oxidase activity in cells was measured as described previously (27). Briefly, MCs grown in serumfree medium containing 5 or 25 mM glucose were washed five times in ice-cold phosphate-buffered saline and were scraped from the plate in the same solution followed by centrifugation at 800 ϫ g, 4°C, for 10 min. The cell pellets were resuspended in lysis buffer. Cell suspensions were homogenized with 100 strokes in a Dounce homogenizer on ice. Aliquots of the homogenates were used immediately to measure NADPHdependent superoxide generation as above.

Measurement of Superoxide Anion Production in Intact Isolated Glomeruli
Measurement of superoxide anion released by isolated glomeruli was performed by detection of superoxide dismutase-inhibitable ferricytochrome c reduction (27,31). Isolated glomeruli were incubated in Hanks' balanced salt solution without phenol red containing 80 M cytochrome c with or without superoxide dismutase (50 g/ml) for 6 h at 37°C. At the end of the incubation, glomeruli were centrifuged for 2 min at 10,000 ϫ g at 4°C. The optical density of the supernatant was measured by spectrophotometry at 550 nm and converted to nmol of cytochrome c reduced using the extinction coefficient ⌬E 550 ϭ 21.0 ϫ 10 3 M Ϫ1 cm Ϫ1 . The reduction of cytochrome c that was inhibitable by pretreatment with superoxide dismutase represents superoxide release.

Detection of Intracellular ROS
The peroxide-sensitive fluorescent probe 2Ј,7Ј-dichlorodihydrofluorescein diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously (27,32). This compound is converted by intracellular esterases to 2Ј,7Ј-dichlorodihydrofluorescein, which is then oxidized by hydrogen peroxide to the highly fluorescent 2Ј,7Ј-dichlorodihydrofluorescein (DCF). Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with ϫ40 Aplanfluo objective and an Olympus fluoview confocal laser-scanning attachment. The DCF fluorescence was measured with an excitation wavelength of 488 nm of light, and its emission was detected using a 510 -550-nm bandpass filter.
Isolated glomeruli were suspended in radioimmune precipitation assay buffer and incubated for 1 h at 4°C. After centrifugation at 10,000 ϫ g for 30 min at 4°C, protein in the supernatant was determined using the Bio-Rad method.
In Vitro Experiments-MCs grown to near confluence were made quiescent by serum deprivation overnight and were exposed to serumfree Dulbecco's modified Eagle's medium containing 5 mM D-glucose, 25 mM D-glucose, or 5 mM D-glucose ϩ 20 mM L-glucose as osmotic control for the specified duration at 37°C. The cells were lysed in radioimmune precipitation assay buffer at 4°C for 30 min. The cell lysates were centrifuged at 10,000 ϫ g for 30 min at 4°C, and protein was determined in the cleared supernatant using the Bio-Rad method.

Determination of Glomerular Surface Area
Light microscopy of hematoxylin and eosin-stained sections from the different treatment groups was used for morphometric studies. The surface area (m 2 ) of a minimum of 50 glomerular sections from each animal was determined in digital images using the Image-Pro Plus 4.5 software (Media Cybernetics). Glomerular surface area was measured in captured digital images by tracing around the perimeter of the glomerular capillary tuft using the polygram tool. The analysis software was calibrated to a stage micrometer.

Immunohistochemistry
Localization of cellular fibronectin was assessed by immunoperoxidase histochemistry using polyclonal Nox4 antibodies or mouse monoclonal antibodies specific for the alternatively spliced extra domain (EIIIA) (clones 3E2, Sigma and IST-9, Serotec, Harlan Bioproducts for Science, Indianapolis, IN) as described previously. Frozen cortical sections (6 m thick) were fixed and permeabilized in acetone for 10 min and then rehydrated in PBS-0.1% BSA for 15 min. Sections were incubated with 0.6% hydrogen peroxide in methanol to block nonspecific peroxidase activity and 0.01% avidin, 0.001% biotin to block localization of endogenous activity before the addition of the appropriate blocking immunoglobulin for 15 min. Sections were incubated with primary antibodies for 30 min in a humidified chamber at room temperature. They were then washed three times in PBS-0.1% BSA and then incubated with biotinylated secondary anti-mouse IgG for 30 min at room temperature. Bound antibody was identified by immunoperoxidase ABC staining following the manufacturer's instructions (Vector Laboratories, Burlingame, CA). The sections were then dehydrated and mounted with Permount (Sigma) and viewed by bright-field microscopy.

Immunofluorescence
Six-m-thick frozen sections were mounted on glass slides and then fixed in acetone. Sections were rehydrated in PBS-0.1% BSA before blocking with the appropriate IgG. Primary antibodies were added at concentrations of 10 g/ml for 1 h at room temperature. After incubation with primary antibodies, sections were washed three times for 5 min in PBS-0.1% BSA. Fluorescence-conjugated secondary antibodies were added at dilutions of 1:100 for 45 min at room temperature followed by washing in PBS-0.1% BSA. Sections were mounted with Crys-  NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 tal Mount (Dako) and allowed to dry before viewing with fluorescence microscopy. ␣-Smooth muscle actin was used as a marker for MCs within the glomerulus.

Statistical Analysis
Results are expressed as mean Ϯ S.E. Statistical significance was assessed by Student's unpaired t test. Significance was determined as probability (p) less than 0.05.

RESULTS
Nox4 Expression-TABLE ONE displays the blood glucose levels and body and kidney weights after 2 weeks of diabetes in the different groups of rats. Untreated diabetic rats and diabetic rats treated with either AS or the corresponding sense Nox4 had equivalently elevated blood glucose concentration at the end of the study period compared with the control rats. Body weight was similarly reduced in the diabetic rats treated with either sense or AS oligonucleotides. Total kidney weight and kidney weight to body weight ratio significantly increased in diabetic rats and sense Nox4-treated diabetic rats compared with nondiabetic control animals. In contrast, total kidney weight in AS Nox4treated diabetic rats was significantly reduced compared with that observed for the diabetic or sense Nox4-treated diabetic groups (TABLE ONE).
To test whether the oligonucleotides were effectively delivered to the kidney and to assess the effect of diabetes on Nox4 expression, we examined the protein levels of Nox4 in renal cortex from the different groups. Western blot analysis using a mouse polyclonal Nox4 antibody directed against recombinant glutathione S-transferase-mouse  showed that a predominant 70-kDa band corresponding to Nox4 was increased in diabetic kidney cortex compared with that in control non- Nox4 and Diabetic Nephropathy diabetic rats. AS Nox4 but not sense Nox4 administration reversed diabetes-induced Nox4 protein expression and significantly reduced Nox4 levels in kidney cortex from diabetic animals (Fig. 1). To confirm the specificity of action of the AS treatment toward Nox4, we also examined the protein expression of another Nox isoform, gp91 phox /Nox2. The levels of gp91 phox /Nox2 were also increased in diabetic animals. More importantly, administration of AS Nox4 had no effect on gp91 phox /Nox2 expression (Fig. 1A). Immunoperoxidase staining showed that Nox4 protein expression is significantly increased in diabetic glomeruli. AS but not sense Nox4 administration markedly reduced diabetes-induced Nox4 protein expression (Fig. 1B). Double immunofluorescence staining revealed the colocalization of Nox4 (green) and ␣-smooth muscle actin (red) in the mesangial area of diabetic glomeruli (Fig. 1C). These observations demonstrate that Nox4 expression is consistent with mesangial distribution. These data indicate that mesangial expression of Nox4 is increased in diabetes and that subcutaneous administration of AS oligonucleotides effectively and specifically inhibits Nox4 NAD(P)H oxidase expression.
ROS Generation-NADPH-dependent superoxide production was significantly increased in renal cortical and glomerular homogenates of diabetic animals compared with controls as measured by lucigeninenhanced chemiluminescence (Fig. 2, A and B). AS Nox4 but not sense Nox4 treatment suppressed diabetes-induced NADPH oxidase activation in cortical and glomerular homogenates (Fig. 2, A and B). Preincubation of homogenates with diphenyleneiodonium, an inhibitor of flavin-containing oxidases, completely blocked NADPH oxidase activity. In addition, superoxide dismutase (50 g/ml) also inhibited the photoemission, thereby confirming identity of the product as superoxide (data not shown). The correlation between the inhibitions of NADPH-dependent ROS generation and the decrease in Nox4 expression following AS Nox4 administration in the diabetic rats suggest that Nox4 is the enzyme responsible for the increase in NADPH oxidase activity in diabetes.
To further confirm the inhibitory effect of AS Nox4 on diabetesinduced oxidative stress in glomeruli, superoxide generation was evaluated ex vivo in isolated glomeruli incubated in the presence of cytochrome c. As shown in Fig. 2C, superoxide generation by isolated glomeruli from diabetic rats was markedly increased compared with control rats. AS Nox4 treatment significantly inhibited the increase in superoxide anion production in diabetic glomeruli. Conversely, superoxide release was not affected by sense Nox4 treatment (Fig. 2C). Effects of Insulin Treatment-To determine whether the increased expression of Nox4 and ROS generation were because of the diabetic state and not because of a toxic effect of STZ, diabetic rats were treated with insulin. Tight glycemic control was achieved in the diabetic rats treated with insulin (mean plasma glucose concentrations on the last day were 102.6 mg/dl Ϯ 3.9 in control rats, 433.5 mg/dl Ϯ 25.8 in diabetic rats, and 103.7 mg/dl Ϯ 32.9 in diabetic rats treated with insulin). Western blot and immunochemical analysis showed that the increased protein levels of Nox4 in diabetic rat kidneys were completely prevented in the rats treated with insulin (Fig. 3, A and B). In addition, the increase in NADPH oxidase activity in cortical homogenates from diabetic animals was also prevented in the diabetic rats treated with insulin (Fig. 3C).
Renal Hypertrophy-As expected, diabetic animals exhibited significantly greater kidney weight to body weight ratio (whole kidney hypertrophy) as compared with nondiabetic rats (TABLE ONE). Treatment with AS Nox4 but not sense Nox4 resulted in a significant decrease in kidney weight (TABLE ONE). These data suggest that Nox4 is involved in renal hypertrophy.
The decrease in whole kidney hypertrophy is accompanied by a decrease in glomerular hypertrophy. Glomerular surface area was

Nox4 and Diabetic Nephropathy
examined and quantified in histological sections of kidneys removed from control, diabetic, AS Nox4-treated, and sense Nox4-treated rats. Fig. 4, A and B, show that glomeruli of diabetic animals are significantly larger compared with controls. AS Nox4 treatment resulted in a decrease in glomerular size. In contrast, glomeruli from sense Nox4treated animals are not different from glomeruli of diabetic animals.
Collectively, these results demonstrate that AS Nox4 treatment reduces whole kidney and glomerular hypertrophy in diabetic animals suggesting that Nox4 is positioned distal to hyperglycemia in the pathway that leads to both whole kidney and glomerular hypertrophy.
Fibronectin Expression-The effect of AS Nox4 administration on the accumulation of the extracellular matrix protein fibronectin was determined in whole cortex and isolated glomeruli by Western blot. As depicted in Fig. 5, A and B, expression of fibronectin protein was significantly increased in both cortex and isolated glomeruli of diabetic animals as compared with controls. The increased expression of fibronectin was markedly reduced in the renal cortex and glomeruli of diabetic rats treated with AS Nox4 (Fig. 5, A and B). Sense Nox4 treatment had no effect on diabetes-induced fibronectin expression.
These observations were confirmed by immunohistochemical analysis of fibronectin expression. As shown in Fig. 5C, the amount of fibronectin was increased in the diabetic group and treatment with AS Nox4 decreased the increased expression of fibronectin induced by diabetes. Sense Nox4 treatment did not alter the enhanced immunoreactivity of fibronectin observed in diabetic rats. Quantitative analysis revealed a significant inhibitory effect of AS Nox4 on glomerular expression of fibronectin induced by diabetes as compared with the control group (Fig. 5D).
Akt/PKB and ERK1/2 Phosphorylation-The serine-threonine kinase Akt/PKB and the mitogen-activated protein kinase family members, ERK1 and -2, are activated by phosphorylation. Both kinases play a critical role in cell growth and hypertrophy as well as matrix expansion (3,27,28,(32)(33)(34)(35)(36)(37)(38)(39). To assess the potential role of these kinases during early DN, the phosphorylation of Akt/PKB and ERK1/2 was examined using phospho-specific antibodies. As illustrated in Fig. 6, phosphorylation of both Akt/PKB and ERK1/2 was markedly increased in the diabetic kidney cortex, and treatment of diabetic animals with AS Nox4 but not sense Nox4 almost abolished this effect. These findings demonstrate that Nox4 is required for diabetes-induced Akt/PKB and ERK1/2 phosphorylation and that the two kinases are positioned downstream of Nox4 in the signaling pathway(s) activated in diabetes.

Effect of High Glucose on ROS Generation and Fibronectin Expression in Cultured
Mesangial Cells-We also assessed the effects of AS Nox4 on ROS production and fibronectin accumulation in cultured rat MCs exposed to high glucose (HG) concentration. The effect of the oligonu-cleotides in MCs was confirmed by the observation that AS Nox4 but not sense Nox4 significantly decreased Nox4 protein expression (Fig.  7A).
AS Nox4-or sense Nox4-transfected MCs were incubated for 24 h in serum-free medium containing either normal glucose concentration (NG, 5 mM D-glucose), HG (25 mM D-glucose), or 5 mM D-glucose ϩ 20 mM L-glucose, and NADPH oxidase activity was measured in crude homogenates using lucigenin-enhanced chemiluminescence. HG caused a robust increase in NADPH-dependent superoxide generation (Fig. 7B). Transient transfection of MCs with AS Nox4 (1 M) but not sense Nox4 (1 M) markedly decreased the activation of NADPH oxidase by HG. To confirm these results, additional studies were undertaken using DCF fluorescence. As shown in Fig. 7C, HG-induced intracellular ROS production was significantly blocked in MCs transfected with AS Nox4. Conversely, fluorescence was not affected by transfection of MCs with sense Nox4.
The effect of HG on fibronectin expression was measured by Western blot analysis in total cell lysates. As anticipated, expression of fibronectin over 24 h was significantly higher in MC cultures that contained 25 mM D-glucose compared with 5 mM D-glucose (Fig. 7D). Similar to ROS generation, transfection of MCs with AS Nox4 significantly decreased HG-mediated increase in fibronectin expression (Fig. 7D). In contrast, sense Nox4 did not alter the increase in fibronectin accumulation in MCs exposed to HG. These effects are not observed in cells cultured with osmotic control. These findings demonstrate that Nox4 NAD(P)H oxidase is involved in HG-stimulated extracellular matrix protein fibronectin production in MCs.

DISCUSSION
Oxidative stress has been implicated in the pathogenesis of diabetic complications (8 -15, 21, 22). However, the mechanisms of ROS generation in diabetes are not fully understood. In this study, we demonstrate that Nox4 is a major source of ROS overproduction in diabetes and that Nox4-derived ROS mediate Akt/PKB and ERK1/2 activation, kidney hypertrophy, and fibronectin expression.
Elevated ROS levels contribute to the development of diabetic vascular complications, such as atherosclerosis and DN (8 -15, 20 -22). In the vasculature, the most important enzyme responsible for ROS production is NAD(P)H oxidase. This oxidase is involved in vascular pathology caused by hypercholesterolemia or hypertension (20 -22). NAD(P)H oxidase was originally found in neutrophils and is composed of the catalytic subunit gp91 phox together with the regulatory subunits p22 phox , p47 phox , and p67 phox and the small GTPase Rac (17,20,40). Electrons from NAD(P)H are transferred through the enzyme to molecular oxygen to generate superoxide and subsequently other ROS such as hydrogen peroxide. Gp91 phox is only one member of a family of homologous proteins termed Nox (17,20). The kidney is known to express NAD(P)H oxidase and generate ROS (12, 15, 17-19, 21, 22). The isoform Nox4 was cloned from the kidney and found to be highly expressed in this organ (17,20,(25)(26)(27). Nox4 is nearly identical in size and structure to gp91 phox (also known as Nox2). However, the requirement for Nox4 activity of other components of the gp91 phox /Nox2 complex is not known. Nox4 is a 578-amino acid protein that exhibits 39% identity to gp91 phox /Nox2 with special conservation in the membrane-spanning regions and binding sites for NADPH, FAD, and heme, the electron transfer centers that pass electrons from NAD(P)H to oxygen to form superoxide (17,20,25,26). We show increased expression of Nox4 protein in the kidney of STZ-induced diabetic rats that is associated with an increase in NADPHdependent ROS generation in kidney cortex and isolated glomeruli. Immunostaining analysis reveals that diabetes up-regulates Nox4 pro-FIGURE 6. Effects of AS Nox4 treatment on Akt/PKB and ERK1/2 activation. ERK1/2 and Akt/PKB phosphorylation was assessed in cortical homogenates using anti-phospho-specific ERK1/2 and Akt/PKB antibodies. Representative results of Western blot analysis were obtained from three independent samples from each group. S, sense. NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 tein levels in a pattern consistent with mesangial cell distribution. The administration of AS Nox4 markedly inhibited diabetes-induced NADPH oxidase activity concomitantly with the down-regulation of Nox4 protein expression. The increases in Nox4 protein expression and ROS generation were prevented by insulin treatment, suggesting that these changes were caused by the diabetic state and were not a direct toxic effect of STZ. Interestingly, gp91 phox /Nox2 was also up-regulated in the diabetic kidney. This is in agreement with previous reports (20,(41)(42)(43) showing that diabetes enhanced expression of gp91 phox /Nox2 in the kidney and vasculature. However, no attenuation of gp91 phox /Nox2 expression was seen in AS Nox4-treated rats, indicating that the decrease in ROS generation is related to inhibition of Nox4. The lack of correlation between ROS generation and increased gp91 phox /Nox2 expression in the AS Nox4-treated animals is somewhat surprising. However, unlike the requirement for Nox4 activation, activation of gp91 phox /Nox2 is dependent on a number of cytosolic and membrane subunits that form the active enzyme complex, and these may not be readily available or not regulated by diabetes. Indeed, there is emerging evidence that in contrast to gp91 phox /Nox2, Nox4 functions independently of the presence of the cytosolic subunits (44,45). Therefore, it is tempting to speculate that Nox4 activity depends primarily on the expression of the catalytic unit itself. A more complete explanation will await a better understanding of the mechanism of Nox4 activation and its precise subunit requirement. In addition, the function of Nox4 as source of ROS in diabetes is supported by the in vitro observation that transfection of MCs with AS Nox4 markedly reduced high glucoseinduced NADPH oxidase activation. The potent effect of AS oligonucleotides is most likely because of their uptake and sequestration in kidney tissue and cells (46).

Nox4 and Diabetic Nephropathy
The nature of the enzymatic sources of oxidative stress in diabetes or upon exposure of cells to high glucose is not precisely defined. Mitochondrial oxidation is an important source of ROS in diabetes (47)(48)(49)(50). Nishikawa et al. (48) emphasized the pivotal role of the increase in production of ROS from the mitochondrial electron transport chain in diabetes. ROS derived from a mitochondrial source were shown to be predominant in various cell types including MCs after exposure to high glucose (48,(51)(52)(53)(54). However, high glucose-induced ROS generation also occurs through activation of a p47 phox -containing NAD(P)H oxidase in cultured cells (55) and in vivo in the aorta of a rat model of type 2 diabetes, characterized by an up-regulation of p22 phox and gp91 phox / Nox2 (43). Likewise, in STZ-induced diabetic rats expression of certain components of the oxidase is augmented in the kidney (13,23,24). It is possible that both enzymatic pathways play a role in diabetes-and high glucose-induced ROS generation. For example, ROS generated by membrane-bound NAD(P)H oxidase may enhance generation of superoxide by mitochondria. The recent findings that mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling support this contention (56). Moreover, Kimura et al. (57) recently reported that Ang II-induced ROS generation via NAD(P)H oxidase triggered mitochondrial ROS release in cardiac myocytes. Conversely, mitochondrial ROS generation may lead to NAD(P)H oxidase activation (58). Such interactions are compatible with the concept of ROS-triggered ROS generation.
Our work not only demonstrated that Nox4-derived ROS contribute to oxidative stress during the initial stages of diabetes but also provided evidence that Nox4-dependent ROS generation mediates renal hypertrophy and fibronectin expression. Inhibition of Nox4 oxidase by administration of AS Nox4 reduced whole kidney hypertrophy and glomerular hypertrophy as well as fibronectin accumulation in diabetic cortex and glomeruli. Furthermore, transfection of cultured MCs with AS Nox4 significantly reduced high glucose-induced accumulation of fibronectin. The mechanisms by which diabetes and high glucose concentrations activate the oxidase remain speculative. They may exert a direct effect on Nox4 to stimulate hypertrophy and fibronectin expression or indirectly via the release of other mediators such as Ang II and/or transforming growth factor-␤. Oxidants may also alter matrix-degrading enzymes. We have shown previously that Ang II induces protein synthesis and hypertrophy via Nox4 in MCs (27,28). Thus, the reninangiotensin system may contribute to the stimulation of Nox4-based NAD(P)H oxidase activity. This hypothesis is supported by the observation that ROS generation and NAD(P)H oxidase subunit p47 phox protein expression were increased in glomeruli of rats with type 1 diabetes, effects that were inhibited by treatment with angiotensin-converting enzyme inhibitor or Ang II type 1 (AT 1 ) receptor blocker (13). High glucose concentration enhances Ang II generation via up-regulation of angiotensinogen, angiotensin-converting enzyme, or renin in renal cells including MCs (59).
We further dissected the involvement of Nox4 by identifying the downstream targets of the oxidase in the signaling cascade linking diabetes and high glucose to cell hypertrophy and fibronectin expression. There is evidence that ERK1/2 mediates hypertrophy and extracellular matrix accumulation both in animal models of diabetes and in cultured renal cells (3,7,36,38,39,60). In vitro studies suggested that activation of the Akt/PKB pathway is involved in renal cell hypertrophy or matrix accumulation (27,32,33,37,61,62). We now demonstrate that both ERK1/2 and Akt/PKB are activated in vivo and that inhibition of Nox4 function with AS Nox4 nearly abrogates diabetes-induced activation of ERK1/2 and Akt/PKB, suggesting that Nox4 functions as an upstream activator of the two kinases not only in vitro but also in vivo. It is tempting to speculate that Nox4 is a pivotal signal transducer commonly shared by both hypertrophic and fibrotic pathways triggered by the diabetic milieu in the kidney.
In conclusion, this study establishes that activation of the NAD(P)H oxidase Nox4 plays a critical role in diabetes-induced oxidative stress, kidney hypertrophy, and fibronectin expression. It would be important to confirm the efficacy of anti-Nox4 therapy in chronic progressive DN. Specific inhibition of this enzyme may selectively target several important biological responses to prevent or reverse pathophysiologic manifestations of diabetes.