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Published online October 24, 2007
Diabetes 57:460-469, 2008
DOI: 10.2337/db07-1119
© 2008 by the American Diabetes Association
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Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product–Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-{alpha}–Dependent Pathway

Vicki Thallas-Bonke1, Suzanne R. Thorpe2, Melinda T. Coughlan1, Kei Fukami1, Felicia Y.T. Yap1, Karly C. Sourris1, Sally A. Penfold1, Leon A. Bach3, Mark E. Cooper1,3, and Josephine M. Forbes1,3

1 Juvenile Diabetes Research Foundation (JDFR) Albert Einstein Centre for Diabetes Complications, Diabetes and Metabolism Division, Baker Medical Research Institute, Melbourne, Victoria, Australia
2 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina
3 Department of Medicine and Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia

Address correspondence and reprint requests to Vicki Thallas-Bonke, JDRF Albert Einstein Centre for Diabetes Complications, Diabetes and Metabolism Division, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Rd., Central, Melbourne, Victoria, Australia, 8008. E-mail: vicki.thallas{at}baker.edu.au

Abbreviations: AGE, advanced glycation end product; CML, carboxymethyllysine; ELISA, enzyme-linked immunosorbent assay; PKC, protein kinsase C; RAGE, receptor for AGE; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE—Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined.

RESEARCH DESIGN AND METHODS—Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg · kg–1 · day–1; weeks 16–32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-{alpha} inhibitor Ro-32-0432.

RESULTS—Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen–associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-{alpha} to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-{alpha} attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin.

CONCLUSIONS—In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-{alpha} is downstream of the AGE–receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.

Diabetic nephropathy, as with other diabetic vascular complications, appears to be multifactorial in origin, involving a number of key pathways, including advanced glycation, activation of intracellular signaling molecules such as protein kinase C (PKC), and increased generation of reactive oxygen species (ROS) (1). However, the nature of the interactions among these various pathways remains uncertain.

While mitochondria are the predominant source of "accidental" oxidative stress in nondisease states during oxidative phosphorylation, cells deliberately use other sources of ROS, from the cytosol for host defense against pathogens or to induce cell signaling. This cytosolic ROS production can result from the activation of various enzymes, including NADPH oxidase, nitric oxide (NO) synthase, and myeloperoxidase, with increasing evidence that NADPH oxidase appears to be the major cytosolic source of ROS generation in diabetes (2). Classically, NADPH oxidase is composed of two membrane-associated components, p22phox and gp91phox, and four major cytosolic components, p47phox, p40phox, p67phox, and rac-1/2. In addition, gp91phox has other homologues that are present within the kidney, namely nox-3, seen in fetal kidney, and nox-4, which is predominately expressed in the renal cortex (3).

The receptor for advanced glycation end products (RAGE) is a pattern recognition inflammatory receptor, which engages a number of ligands, including advanced glycation end products (AGEs). AGEs are formed when certain, free amino groups on proteins become permanently modified by reducing glucose, facilitated in diabetes by hyperglycemia and oxidative stress (4). Both the accumulation of AGEs (5–7) and RAGE (8,9) expression play important roles in the pathogenesis of diabetic nephropathy. In addition, engagement of RAGE by AGEs induces signal transduction and generates cellular oxidative stress via activation of NADPH oxidase in endothelial cells (10).

Specific upregulation of the subunits of NADPH oxidase, including p47phox (11), nox-4 (12), and p22phox (13) have been demonstrated in diabetes within the kidney. In addition, the inhibitor of NADPH oxidase, apocynin, improves renal function in streptozotocin-induced diabetic rats, albeit over the relatively short period of 4 weeks (3). This agent's postulated mode of action is to impede the recruitment of the p47phox and p67phox subunits to the NADPH oxidase complex (14,15).

The cytosolic generation of ROS has been demonstrated in vitro through activation of RAGE in both proximal tubular and mesangial cells in association with specific activation of NADPH oxidase (16,17). Furthermore, the AGE-RAGE interaction enhances production of the cytokine vascular endothelial growth factor (VEGF) (9), which is postulated to be directly induced by NADPH oxidase and is linked to the pathogenesis of albuminuria in diabetes (18).

Therefore, this study initially aimed to test the utility of the NADPH oxidase inhibitor apocynin in established experimental diabetic nephropathy. This series of experiments addressed AGE-RAGE–induced activation of NADPH oxidase via PKC-{alpha} phosphorylation and its downstream signaling events, including generation of cytosolic ROS, induction of VEGF, and accumulation of basement membrane proteins. Based on these in vivo findings, a series of complementary in vitro studies were performed that further examined the sequence of intracellular events resulting from the AGE-RAGE interaction.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animal model.
Experimental diabetes was induced in male Sprague Dawley rats (200–250 g) by injection of the β-cell toxin streptozocin (50 mg/kg) following an overnight fast. Animals with plasma glucose concentrations in excess of 15 mmol/l, 1 week postinduction of diabetes, were included in the study. Sham-injected control animals (sodium citrate buffer, pH 4.5) were followed concurrently. Diabetic and control animals were randomized into groups (n = 12) that received 1) no treatment (groups D and C) and 2) the NADPH oxidase inhibitor apocynin (15 mg/kg 4'-hydroxy-3'-methoxy-acetophenone; Merck, Whitehouse Station, NJ) by oral gavage from week 16 to 32 (groups CApo and DApo) as a late intervention. Delayed intervention was used (19) to assess the role of such a therapeutic approach with animals that already had evidence of renal disease, albeit not very advanced. It was considered that this strategy was analogous to the human context, in which treatment is generally started in diabetic subjects once they already have some features of diabetic nephropathy such as microalbuminuria. Two to three units of Ultralente insulin (Ultratard HM; Novo Industries, Bagsvaerd, Denmark) were administered daily to diabetic animals to prevent ketoacidosis and improve survival. Body weight, mean systolic blood pressure by tailcuff plethysmography (20), glomerular filtration rate using 99Tc-DTPA (21), albumin excretion rate by a rat albumin enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Montgomery, TX), and glycated hemoglobin (22) were measured every 8 weeks. All animal procedures were in accordance with guidelines set by the Alfred Medical Research and Education Precinct Ethics Committee and the National Health and Medical Research Council of Australia.

Cell culture.
Mesangial cells were isolated from rat kidneys as previously described (23). Mesangial cells were grown in 10-cm2 tissue culture plates in DMEM containing 10% FCS (fetal bovine serum) and 5.5 mmol/l D-glucose. After 24 h, incubation medium was replaced with DMEM containing high glucose (25 mmol/l) concentrations in 2% fetal bovine serum with appropriate treatments and incubated for 7 days. AGE-modified bovine serum albumin (AGE-BSA) was added to some groups at 100 µg/ml as previously described (24). Endotoxin was removed from AGE-BSA preparations by Detoxigel columns (Pierce Biotechnology, Rockford, IL). Endotoxin content of subsequent eluates was assessed via Limulus amoebocyte lysate validity testing (AMS Laboratories, Sydney, Australia). There was no detectable endotoxin within the filtered samples. Cells were harvested as outlined below. Ro-32-0432 (Calbiochem, Darmstadt, Germany) is a selective cell permeable PKC inhibitor, which displays a 10-fold greater selectivity for PKC-{alpha} over other PKC isoforms and was used at a concentration of 10 nmol/l. Apocynin was used at a concentration of 1 µmol/l. L-glucose (25 mmol/l) was used as an osmotic control.

Histological assessment of kidney injury.
Kidney sections were stained with periodic scid Schiff's stain for quantitation of glomerulosclerosis. The degree of glomerulosclerosis, which was defined as thickening of the basement membrane and mesangial expansion, was evaluated by a semiquantitative method as described previously (19). In brief, 2-µm kidney sections were stained with periodic acid Schiff's stain and observed under a light microscope in a masked fashion at a magnification of x400 using the Analysis Imaging System (Imaging Research, St. Catherines, ON, Canada). Forty glomeruli in each kidney were graded in accordance with their severity of glomerular damage (0, normal; 1, slight glomerular damage, the mesangial matrix and/or hyalinosis with focal adhesion involving <25% of the glomerulus; 2, sclerosis of 26–50%; 3, sclerosis of 51–75%; and 4, sclerosis of >75% of the glomerulus). The glomerulosclerotic indexes were calculated using the following formula: glomerulosclerotic index = (1 x n1) + (2 x n2) + (3 x n3) + (4 x n4)/no + n1 + n2 + n3 + n4, where nx is number of glomeruli in each grade of glomerulosclerosis (19,25).

Tubulointerstitial area was evaluated using the point-counting technique and performed in the renal cortex of each animal following established methods (26). In each field, 81 points were counted on a 1-cm2 eyepiece graticule with nine equidistant grid lines. A total of 10 high-power fields (x200) per section were counted for each animal in all groups in the corticomedullary area. Percent fractional area was calculated as follows: % fractional area = (number of positive tubulointerstitial grid intersections/total number of grid intersections) x 100.

Carboxymethyllysine ELISA.
Carboxymethyllysine (CML) concentrations in serum and renal cytosolic extracts were assessed by an in-house indirect ELISA (27). Samples (diluted 1:10,000 to 1:20,000 for serum and 1:1,000 for kidney cytosolic) or standard were diluted in 50 mmol/l carbonate buffer (pH 9.6). Results are expressed in nmol · mol lysine–1 · mg–1 protein as compared with a standard, measured by gas chromatography–mass spectrometry (27).

Isolation of skin collagen and analysis of AGEs/advanced lipoxidation end products.
Rat skin collagen was prepared as described previously (19). N{varepsilon}(carboxymethyl)lysine (CML) was quantified by isotope dilution, selected ion-monitoring gas chromatography–mass spectrometry (19) and normalized to their parent amino acid lysine.

Superoxide production.
Kidneys were rapidly excised, placed in oxygen-saturated Krebs buffer, and cut into ~1-mm3 segments (27). The rate of superoxide anion formation in the kidney was determined by chemiluminescence of lucigenin (bis-N-methylacridinium nitrate; Sigma Chemical) using a microtiter plate-reading luminometer (Microlumat Plus; Berthold Technologies, Wildbad, Germany).

PKC activity.
Kidney cortex was homogenized in ice-cold buffer A (20 mmol/l Tris-HCl, pH 7.5; 2 mmol/l EDTA; 10 mmol/l EGTA; and 0.25 mol/l sucrose) containing complete protease (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitors (Sigma, St. Louis, MO). The homogenates were centrifuged at 1,000g for 10 min. These supernatants were then centrifuged at 10,000g at 4°C for 20 min. The supernatants were then ultracentrifuged at 100,000g for 1 h at 4°C. This supernatant was retained as the cytosolic fraction, and the pellet was resuspended in buffer B (buffer A with 1% Triton X-100) and ultracentrifuged at 100,000g for 1 h at 4°C. This supernatant was retained as the membranous fraction (28). Membrane and cytosolic fractions were assayed for PKC activity using the StressXpress PKC Kinase Activity Assay Kit (EKS-420A; Stressgen Bioreagents, Victoria, BC, Canada) as described previously (27).

Western immunoblotting.
Membrane fractions prepared for the PKC activity assay above were used for Western immunoblotting. Mesangial cells were sonicated and spun at 400g for 5 min. The supernatant was collected and the protein content was measured using the BCA protein assay (Perbio Science UK, Cheshire, U.K.).

For Western blotting (6), 5 µg of membranous samples and 15 µg of mesangial cell supernatants were incubated at 95°C for 3 min and separated on an 8% SDS-PAGE and transferred onto polyvinylidine fluoride membranes. Nonspecific binding sites were blocked for 1 h with 5% (wt/vol) nonfat milk powder in TBS followed by overnight incubation of primary antibodies, rabbit anti–PKC-{alpha} (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–phospho PKC-{alpha} (1:1,000; Upstate, Lake Placid, NY), and mouse monoclonal β-actin (1:5,000; Abcam, Cambridge, U.K.). Bound antibodies were amplified and detected by reaction with an Amersham ECL kit (Amersham Biosciences, Piscataway, NJ). The band intensity was quantified using a microcomputer imaging device and expressed as (density times area) minus background.

VEGF ELISA.
The Quantikine Mouse ELISA kit (R&D Systems, Minneapolis, MN) was used to measure VEGF, in an aliquot of urine from the 24-h collection and serum. The cytosolic and membranous kidney samples obtained for the PKC activity assay described above were also used for the measurement of VEGF, as was the media collected from the culture of mesangial cells, which was concentrated before quantitation. The values of urinary and serum VEGF are expressed as picograms per millimole of creatinine as determined by an autoanalyzer (Alfred Hospital, Melbourne, Australia) (27).

Immunohistochemistry.
Immunohistochemistry was performed as previously described (19). In brief, slides for fibronectin and collagen IV were digested with 0.4% pepsin for 4 min at 37°C. Slides for p47phox required no pretreatment. The primary antibodies used were rabbit anti-human fibronectin (1:4,000; DakoCytomation, Carpentaria, CA), goat anti-human collagen IV (1:500; Southern Biotech, Birmingham, AL), and rabbit anti-p47phox (1:400; Upstate). Quantitation of renal cortical immunohistochemistry was performed by computer-aided densitometry (Optimus 6.5; Media Cybernetics, Silver Spring, MD), as previously described (19).

ROS production in primary mesangial cells.
Hydrogen peroxide production in primary rat mesangial cells was determined using CM-H2DCFDA (5', 6'-chloromethyl-2',7' dichlorodihydro-fluorescein diacetate) (Molecular Probes, Eugene, OR). Mesangial cells were seeded onto 12-well plates at a concentration of 5 x 104 per well in 10% serum media and exposed to 100 µg/ml AGE-BSA or BSA with or without 1 µmol/l apocynin. After incubation for 3 days, serum-starved cells were exposed to 5 µmol/l CM-H2DCFDA for 10 min at 37°C. After 10 min, the cells were washed twice with serum-free media and images were collected using a Zeiss LSM 510 META confocal microscope (23).

Flow cytometric analysis of surface RAGE expression on primary mesangial cells.
Primary mesangial cells were grown for 7 days as outlined above in the cell culture section. Mesangial cells were washed in sterile PBS and centrifuged 1,000 rpm for 5 min and counted in a hemocytometer. A total of 5 x 105 cells were stained in each tube with 15 µl of the primary antibody, goat anti-RAGE (Santa Cruz Biotechnology), in the dark for 30 min. Cells were then washed with excess buffer (PBS with 2.5% fetal bovine serum and 0.01% NaN3), and fluorescein isothiocyanate–conjugated rabbit anti-goat IgG, F(ab')2 (1:100, Chemicon International, Temecula, CA), was added as the secondary antibody. Following a second 30-min incubation in the dark, the cells were washed again in excess buffer. A minimum of 10,000 cells were collected on FACsCalibur (BD Biosciences, Lane Cove, NSW, Australia) and the data analyzed using WinMDI 2.8. RAGE-positive cells were identified relative to the appropriate isotopic controls.

Statistical analysis.
Results are expressed as means ± SD, unless otherwise specified. Data for albuminuria were not normally distributed and were therefore analyzed following logarithmic transformation and are expressed as geometric mean ± tolerance factors. Analyses were performed by ANOVA followed by post hoc analysis using Tukey's test. A value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physiological and structural parameters.
Diabetic rodents demonstrated a significant elevation in plasma glucose concentrations and glycated hemoglobin, which was unaffected by apocynin treatment (Table 1). Diabetes was also associated with decreased body weight. The 24-h albumin excretion rate was increased with diabetes and reduced significantly by apocynin therapy (Table 1). Although diabetes induced increases in albuminuria at week 16 (C group 1.7 x/÷ 1.5 mg/24 h; D group 14.1 x/÷ 2.3 mg/24 h; P < 0.01 with geometric mean x/÷ tolerance factors shown), there were no differences seen between the untreated D and DApo groups at that time (DApo group, 13.1 x/÷ 2.1 mg/24 h; P > 0.05 vs. D group). Thus there were no differences in albumin excretion rate among the diabetic groups before the commencement of treatment at week 16. Mean systolic blood pressure was increased with diabetes, but no statistically significant change was noted with apocynin treatment. Glomerular filtration rate corrected for body weight was increased with diabetes, which was not attenuated with apocynin treatment (Table 1). The glomerulosclerotic index and tubulointerstitial area were increased with diabetes, and both markers of renal structural injury were attenuated by apocynin treatment (Table 1).


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  		TABLE 1 Physiological and structural parameters for all rodents at week 32

 
The accumulation of tissue and circulating AGEs are unaffected by NADPH oxidase.
Circulating levels of the nonfluorescent AGE and CML were increased significantly with diabetes but were not affected by apocynin treatment (Fig. 1A). In addition, cytosolic CML levels in renal cortices from diabetic rats were also elevated (Fig. 1B), but no changes were seen with apocynin therapy (Fig. 1B). Furthermore, diabetic rats had increased concentrations of skin collagen–associated CML, and, again, this parameter was not affected by apocynin treatment (Fig. 1C).


Figure 1
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  		FIG. 1. CML (a representative AGE) accumulation at week 32 (n = 10/group). A: Serum CML concentrations determined by ELISA. B: Renal cytosolic accumulation of CML as determined by ELISA. C: Skin collagen–associated CML levels by gas chromatography/mass spectrometry. *P < 0.001 vs. control group. (A high-quality digital representation of this image can be found at http://dx.doi.org/10.2337/db07-1119.)

 
Renal cytosolic superoxide generation and activation of PKC-{alpha} are prevented by blockade of NADPH oxidase.
Lucigenin-enhanced superoxide generation by NADPH oxidase in the cytosol of renal cortices was increased by >300% in diabetic rats and totally prevented by apocynin treatment (Fig. 2A). Immunohistochemical staining of the NADPH oxidase isoform, p47phox, in kidney revealed widespread staining in the glomeruli. The expression of glomerular p47phox was increased with diabetes and ameliorated by treatment with apocynin (Fig. 2B and C [D–F]). PKC activity in renal membrane fractions isolated from diabetic rats was increased by ~100% (Fig. 3A) compared with control rats and was reversed by apocynin. There was no change in the activity of PKC within the cytosol among the various groups (Fig. 3B). The ratio of phosphorylated PKC-{alpha} to total PKC-{alpha} was also increased in renal cortical membranes from diabetic rats (Fig. 3C and D), and this was normalized in the apocynin group.


Figure 2
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  		FIG. 2. Renal cytosolic superoxide production (n = 10/group). A: Lucigenin enhanced NADPH oxidase–dependent superoxide production. *P < 0.05 vs. control group; {dagger}P < 0.01 vs. diabetic group. B: Computer-aided analysis of renal glomerular p47phox at 32 weeks, expressed as percent area. {ddagger}P < 0.001 vs. control group; §P < 0.0001 vs. diabetic group. C: Periodic scid Schiff's staining (A–C) and immunostaining of the NADPH oxidase isoform p47phox in kidney glomeruli of 32-week-old rats (D–F). A and D represent control kidney; B and E represent diabetic kidney; D and F represent diabetic plus apocynin-treated kidney (x400 magnification).

 

Figure 3
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  		FIG. 3. Renal cortical PKC activity (n = 10/group). A: Membranous PKC activity by ELISA. B: Cytosolic PKC activity by ELISA. C: Quantitation of immunoblots stained for renal cortical phosphorylated PKC-{alpha} (82 kDa) and total PKC-{alpha} (80 kDa) at 32 weeks. The graph represents the densitometric analysis (means ± SE) of individual Western blots expressed as relative optical density. D: Representative immunoblots stained for phosphorylated PKC-{alpha} (82 kDa; A) and PKC-{alpha} (80 kDa; B) in kidney membranes at 32 weeks. *P < 0.001 vs. control group; {dagger}P < 0.001 vs. diabetic group; {ddagger}P < 0.05 vs. control group; §P < 0.01 vs. diabetic group.

 
The PKC-{alpha}–dependent cytokine VEGF is decreased following therapy to reduce NADPH oxidase activity.
Diabetes was associated with significant elevations in urinary (Fig. 4A) and serum (Fig. 4B) VEGF concentrations after 32 weeks. These diabetes-induced increases in urine and serum VEGF were ameliorated by apocynin therapy. Membrane, but not cytosolic, fractions from diabetic renal cortices also demonstrated elevations in VEGF (Fig. 4C and D) when compared with the appropriate control groups. Apocynin therapy attenuated the increase in membranous VEGF seen in diabetic kidneys.


Figure 4
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  		FIG. 4. Protein expression of the PKC-{alpha}–dependent profibrotic cytokine VEGF (n = 10/group). A: Urinary VEGF at 32 weeks expressed as pg/mmol creatinine. B: Serum VEGF at 32 weeks expressed as pg/mmol creatinine. C: Renal cortical membranous VEGF at 32 weeks. D: Renal cortical cytosolic VEGF at 32 weeks. *P < 0.01 vs. control group; {dagger}P < 0.01 vs. diabetic group; {ddagger}P < 0.05 vs. control group.

 
Diabetes-induced increases in extracellular matrix proteins are not seen in diabetic rats treated with an NADPH oxidase inhibitor.
The expression of renal cortical fibronectin was significantly increased in diabetic kidneys and reduced by apocynin (Fig. 5A and C [A–C]). In addition, renal cortical expression of collagen IV was increased almost twofold with diabetes and was attenuated by apocynin (Fig. 5B and C [D–F]).


Figure 5
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  		FIG. 5. Morphometric determination of immunohistochemistry of extracellular matrix proteins (n = 10/group). A: Computer-aided analysis of renal glomerular fibronectin at 32 weeks expressed as percent area. B: Computer-aided analysis of renal glomerular collagen IV at 32 weeks expressed as percent area. C: Representative photomicrographs of fibronectin (A–C) and collagen IV (D–F) immunostaining. A and D represent control kidney; B and E represent diabetic kidney; C and F represent diabetic plus apocynin kidney (x200 magnification). *P < 0.001 vs. control group; {dagger}P < 0.01 vs. diabetic group; {ddagger}P < 0.001 vs. diabetic group. (A high-quality digital representation of this image can be found at http://dx.doi.org/10.2337/db07-1119.)

 
Primary mesangial cells exposed to AGEs generate cytosolic ROS via an NADPH oxidase and PKC-{alpha}–dependent pathway.
Previous studies by our group showed that AGE blockade reduced VEGF expression via PKC-{alpha} (6), and in this study NADPH blockade also appeared to affect this signaling molecule in a similar manner. However, since NADPH oxidase blockade does not influence AGEs it was postulated that NADPH oxidase activation, as had been suggested in other studies, is downstream of the AGE/RAGE interaction. Therefore, in vitro studies with mesangial cells were performed to further characterize this pathway.

The ratio of phosphorylated PKC-{alpha} to total PKC-{alpha} in primary mesangial cells exposed to high glucose in the presence of AGE-BSA was elevated compared with controls (Fig. 6A) and BSA exposed cells (data not shown) (P = 0.006). Both apocynin and Ro-32-0432 significantly reduced this AGE-induced elevation in the phosphorylated PKC-{alpha}–to–PKC-{alpha} ratio. These results are consistent with those seen with respect to activity of PKC. Specifically, PKC activity was increased by concomitant AGE exposure and attenuated by both apocynin and Ro-32-0432 (Fig. 6B).


Figure 6
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  		FIG. 6. AGE-mediated effects on cultured primary mesangial cells (n = 6/group). Experiments were performed for 7 days in 25 mmol/l glucose in the presence and absence of AGE-BSA (100 µg/ml). APO, apocynin (NADPH oxidase inhibitor 1 µmol/l; Ro-32-0432 [PKC-{alpha} inhibitor 10 nmol/l]); HG, high glucose. A: Phosphorylated PKC-{alpha}–to–total PKC-{alpha} ratio. B: Cellular PKC activity by ELISA. C: Secreted VEGF in concentrated cell culture media. D: Flow cytometric analysis of cell surface RAGE. E: Cytosolic ROS generation with the fluorophore CM-H2DCFDA. *P < 0.05 vs. HG; {dagger}P < 0.001 vs. HG + AGE; {ddagger}P < 0.01 vs. HG; §P < 0.01 vs. HG + AGE; #P < 0.05 vs. HG + AGE; **P < 0.001 vs. HG + AGE; {dagger}{dagger}P < 0.001 vs. HG.

 
The concentration of VEGF secreted into the cell culture media was increased by incubation with AGE-BSA when compared with controls (Fig. 6C). Furthermore, these AGE-induced elevations were ameliorated by treatment with either apocynin or Ro-32-0432 (Fig. 6C). Cell surface expression of RAGE in cells exposed to high glucose in the presence of AGE-BSA was increased when compared with control cells. This increase in RAGE expression was ameliorated by treatment with either apocynin or Ro-32-0432 (Fig. 6D). Cytosolic ROS was higher in primary mesangial cells that were treated with AGE-BSA for 7 days when compared with treatment with BSA alone (Fig. 6E). Both blockade of NADPH oxidase with apocynin or inhibition of PKC-{alpha} activity with Ro-32-0432 ameliorated the elevations in cytosolic ROS generated by AGE exposure (Fig. 6E).


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study extends the therapeutic potential of blockade of NADPH oxidase in experimental diabetic nephropathy. Previously, Asaba et al. (29) have shown that apocynin reduced proteinuria and retarded mesangial expansion in an experimental study over the period of 4 and 8 weeks. In the present study, more prolonged apocynin administration was commenced (from week 16) once early renal changes, such as microalbuminuria, were already evident. This was associated with similar renal functional and structural benefits (Table 1, Fig. 2C [A–C]), with apocynin specifically reducing diabetes-associated albuminuria, glomerulosclerosis, and tubulointerstitial injury. Blockade of NADPH oxidase to prevent progressive diabetic nephropathy, as assessed in the current study design, has important clinical implications, since renoprotective therapies in humans are not generally administered at the time of diagnosis of diabetes but usually after some renal dysfunction, such as microalbuminuria, is already evident. Although apocynin is not suitable for human use, the present study suggests that blockade of NADPH oxidase is a valid intervention to consider for combating established diabetic nephropathy. Furthermore, to our knowledge this is the first study to look at the long-term effect of apocynin administration, since previous studies have only been performed for up to 8 weeks’ duration. This therapeutic strategy warrants further investigation, since other novel compounds other than apocynin that block NADPH oxidase with as-yet unknown toxicity profiles in humans are currently in clinical development and could ultimately have a role in human diabetic nephropathy. Furthermore, with increasing availability of animals where Nox isoforms (30) have been specifically targeted, it is now feasible to consider mechanistic studies in such models.

In the present in vivo study, we have shown an increase in renal cytosolic generation of superoxide, which is ameliorated by apocynin, indicating a role for NADPH oxidase. This was seen in the context of increased glomerular expression of the NADPH oxidase subunit p47phox in diabetic kidneys, which was ameliorated by treatment with apocynin as well as translocation of phosphorylated PKC-{alpha} to diabetic renal membranes in association with PKC activation. Indeed, previous studies in rodent aortas have shown that vascular superoxide is normalized by a PKC inhibitor and that the major source of superoxide was indeed NADPH oxidase (31). Interestingly, our results indicate that accumulation of both tissue and circulating AGEs are unaffected by NADPH oxidase in this model, as apocynin did not attenuate the diabetes-induced increases in these parameters. The expression of RAGE, although induced by experimental diabetes or AGE-BSA in vitro, was ameliorated by blockade of NADPH oxidase or PKC-{alpha}. Furthermore, our in vitro studies in primary mesangial cells showed that cellular ROS generation was induced by exposure to AGE-BSA and was ameliorated with apocynin or blockade of PKC-{alpha}.

Previously, it has been suggested that phosphorylation of the NADPH oxidase subunit p47phox is required for assembly of this complex. In the present study, there is increased expression of renal glomerular p47phox seen with diabetes, which was ameliorated with apocynin. In neutrophils, NADPH oxidase is activated via PKC-mediated phosphorylation of the p47phox subunit (32). In vivo, renal translocation and activation of a number of PKC isoforms, including PKC-{alpha}, have been reported in various models of experimental diabetic nephropathy (33–35). In addition, mice with a genetic deletion of PKC-{alpha} are protected against the development of albuminuria and overt diabetic nephropathy (36). We have previously shown that AGE-RAGE interactions induce PKC-{alpha} in diabetic rats (6). In addition, in the present study, PKC-{alpha} inhibition normalized AGE-induced cellular ROS generation. However, it should be noted that increases in the ratio of phosphorylated to total PKC-{alpha} were ameliorated by NADPH oxidase inhibition in vivo, and these findings were confirmed in vitro with NADPH oxidase inhibition attenuating mesangial cell increases in PKC-{alpha} phosphorylation induced by AGEs. It is important to note that sustained activation of PKC is also mediated by ROS (37). Therefore, the effects of apocynin on PKC activity and phosphorylated PKC-{alpha} expression may be via the inhibition of renal cellular ROS generation, in an ongoing feedback loop involving phosphorylation of p47phox.

PKC-{alpha} is a key regulator of VEGF expression, particularly in the setting of hyperglycemia, since diabetes in PKC-{alpha}–deficient mice does not induce increased renal VEGF expression (36). Furthermore, our own group has shown that decreasing PKC-{alpha} activation with the AGE-lowering therapy alagebrium (ALT-711) results in decreased VEGF expression (6). Indeed, we postulated in that study and confirmed in vascular smooth muscle cells in vitro that alagebrium is, in fact, a direct inhibitor of the activity of PKC-{alpha}. Therefore, it is not surprising in the present study that accumulation of AGEs appears to be upstream of PKC activation. This is further supported by our in vitro studies in which blockade of either NADPH oxidase or PKC-{alpha} resulted in less secreted VEGF in the cell culture medium of AGE-treated primary mesangial cells. Furthermore, these studies confirm that the significant increases in VEGF in urine, serum, and renal membranes of diabetic rats are ameliorated by NADPH oxidase blockade. Although albuminuria in diabetes is likely to occur as a result of a number of pathways, VEGF is increasingly considered to play an important role. Indeed, neutralizing antibodies to VEGF have been shown to decrease albuminuria in both short- and long-term diabetes (8,38), and, more recently, a tyrosine kinase inhibitor that targets the VEGF receptor has been shown to confer similar benefits on albuminuria (39). Therefore, it is possible that the antialbuminuric effect of apocynin is related, at least in part, to reduced renal VEGF expression.

A number of studies have implicated AGE accumulation and PKC activation in diabetes complications (35) by identifying a link between these two pathways as well as being critical in downstream expression of extracellular matrix proteins. Indeed, fibronectin deposition is known to result from activation of either pathway (6,40,41). Within the current study, inhibition of NADPH oxidase with subsequent reductions in cytosolic superoxide and PKC-{alpha} activation ameliorated the diabetes-associated increases in renal fibronectin expression. Collagen IV is another extracellular matrix protein influenced by PKC-dependent pathways (42,43) and is also linked to increased ROS generation (44,45). Furthermore, Xia et al. (46) reported that mesangial cell ROS generation by NADPH oxidase in high glucose is dependent on conventional PKC-{alpha} and -β, with these isoforms also required for the increased expression of collagen IV (9,46). In the present study, the increased expression of collagen IV, as well as expansion of the tubulointerstitium in diabetic kidneys, was also reduced by blockade of NADPH oxidase, an effect that has been previously reported by our group following AGE-lowering therapy with alagebrium (6) and aminoguanidine (47). In addition, previous in vitro studies (40,48) demonstrated that blockade of NADPH oxidase in various renal cell types leads to reduced expression of extracellular matrix proteins such as fibronectin.

This series of experiments has investigated the efficacy of therapies that interfere with NADPH oxidase–dependent ROS generation, which is thought to result from AGE-RAGE interactions in the diabetic kidney. Although the accumulation of AGEs remained unaffected by NAPDH oxidase blockade, important downstream pathways implicated in the pathogenesis of diabetic nephropathy, namely RAGE expression, cytosolic ROS generation, PKC activation, VEGF secretion, and extracellular matrix accumulation, were normalized by this intervention (Fig. 7). Nevertheless, one cannot exclude that other effects of Nox inhibition, including systemic effects such as improvement in endothelial function, may also participate in conferring end-organ protection (49). With increasing elucidation of the key interactions between NADPH oxidase and the critical pathways implicated in the progression of diabetic nephropathy, it appears likely that this enzyme is an appropriate therapeutic target to investigate further in human diabetic nephropathy.


Figure 7
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  		FIG. 7. Speculative schema for the involvement of NADPH oxidase in progressive diabetic nephropathy.

 


   ACKNOWLEDGMENTS
 
This work was supported by the Juvenile Diabetes Research Foundation (JDRF), the National Health and Medical Research Council of Australia, and U.S. Public Health Service Grant NIDDK-19971. J.M.F. is a JDRF Career Development Awardee.

The authors thank Gavin Langmaid for his expert care of the animals and Maryann Arnstein for her technical support throughout the study.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 24 October 2007. DOI: 10.2337/db07-1119.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication August 8, 2007 and accepted in revised form October 18, 2007


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001[Medline]
  2. Hancock JT, Desikan R, Neill SJ: Role of reactive oxygen species in cell signalling pathways. Biochem Soc Trans 29:345–350, 2001[Medline]
  3. Gill PS, Wilcox CS: NADPH oxidases in the kidney. Antioxid Redox Signal 8:1597–1607, 2006[Medline]
  4. Fu MX, Wells-Knecht KJ, Blackledge JA, Lyons TJ, Thorpe SR, Baynes JW: Glycation, glycoxidation, and cross-linking of collagen by glucose: kinetics, mechanisms, and inhibition of late stages of the Maillard reaction. Diabetes 43:676–683, 1994[Abstract]
  5. Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G: Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fluorescence in streptozocin-induced diabetic rat. Diabetes 40:1328–1334, 1991[Abstract]
  6. Thallas-Bonke V, Lindschau C, Rizkalla B, Bach LA, Boner G, Meier M, Haller H, Cooper ME, Forbes JM: Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via a protein kinase C-{alpha}–dependent pathway. Diabetes 53:2921–2930, 2004[Abstract/Free Full Text]
  7. Vlassara H, Striker LJ, Teichberg S, Fuh H, Li YM, Steffes M: Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc Natl Acad Sci U S A 91:11704–11708, 1994[Abstract/Free Full Text]
  8. Flyvbjerg A, Denner L, Schrijvers BF, Tilton RG, Mogensen TH, Paludan SR, Rasch R: Long-term renal effects of a neutralizing RAGE antibody in obese type 2 diabetic mice. Diabetes 53:166–172, 2004[Abstract/Free Full Text]
  9. Wendt TM, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, Bucciarelli LG, Rong LL, Moser B, Markowitz GS, Stein G, Bierhaus A, Liliensiek B, Arnold B, Nawroth PP, Stern DM, D'Agati VD, Schmidt AM: RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy. Am J Pathol 162:1123–1137, 2003[Abstract/Free Full Text]
  10. Kislinger T, Fu C, Huber B, Qu W, Taguchi A, Du Yan S, Hofmann M, Yan SF, Pischetsrieder M, Stern D, Schmidt AM: N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem 274:31740–31749, 1999[Abstract/Free Full Text]
  11. Onozato ML, Tojo A, Goto A, Fujita T, Wilcox CS: Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 61:186–194, 2002[Medline]
  12. Li JM, Shah AM: ROS generation by nonphagocytic NADPH oxidase: potential relevance in diabetic nephropathy. J Am Soc Nephrol 14 (Suppl. 3):S221–S226, 2003
  13. Matsunaga-Irie S, Maruyama T, Yamamoto Y, Motohashi Y, Hirose H, Shimada A, Murata M, Saruta T: Relation between development of nephropathy and the p22phox C242T and receptor for advanced glycation end product G1704T gene polymorphisms in type 2 diabetic patients. Diabetes Care 27:303–307, 2004[Abstract/Free Full Text]
  14. Hart BA, Simons JM: Metabolic activation of phenols by stimulated neutrophils: a concept for a selective type of anti-inflammatory drug. Biotechnol Ther 3:119–135, 1992[Medline]
  15. Meyer JW, Schmitt ME: A central role for the endothelial NADPH oxidase in atherosclerosis. FEBS Lett 472:1–4, 2000[Medline]
  16. Doublier S, Salvidio G, Lupia E, Ruotsalainen V, Verzola D, Deferrari G, Camussi G: Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes 52:1023–1030, 2003[Abstract/Free Full Text]
  17. Lal MA, Brismar H, Eklof AC, Aperia A: Role of oxidative stress in advanced glycation end product-induced mesangial cell activation. Kidney Int 61:2006–2014, 2002[Medline]
  18. Abid MR, Tsai JC, Spokes KC, Deshpande SS, Irani K, Aird WC: Vascular endothelial growth factor induces manganese-superoxide dismutase expression in endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism. Faseb J 15:2548–2550, 2001[Free Full Text]
  19. Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC, Jerums G, Cooper ME: The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes. Faseb J 17:1762–1764, 2003[Abstract/Free Full Text]
  20. Ido Y, Chang KC, Lejeune WS, Bjercke RJ, Reiser KM, Williamson JR, Tilton RG: Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C. Microcirculation 8:251–263, 2001[Medline]
  21. O'Brien RC, Allen TJ, Cooper ME, Bach L, Jerums G: Glomerular filtration rate in early experimental diabetes. J Diabet Complications 2:8–11, 1988[Medline]
  22. Cefalu WT, Wang ZQ, Bell-Farrow A, Kiger FD, Izlar C: Glycohemoglobin measured by automated affinity HPLC correlates with both short-term and long-term antecedent glycemia. Clin Chem 40:1317–1321, 1994[Abstract/Free Full Text]
  23. Fukami K, Ueda S, Yamagishi S, Kato S, Inagaki Y, Takeuchi M, Motomiya Y, Bucala R, Iida S, Tamaki K, Imaizumi T, Cooper ME, Okuda S: AGEs activate mesangial TGF-beta-Smad signaling via an angiotensin II type I receptor interaction. Kidney Int 66:2137–2147, 2004[Medline]
  24. Oldfield MD, Bach LA, Forbes JM, Nikolic-Paterson D, McRobert A, Thallas V, Atkins RC, Osicka T, Jerums G, Cooper ME: Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE). J Clin Invest 108:1853–1863, 2001[Medline]
  25. Ji H, Pesce C, Zheng W, Kim J, Zhang Y, Menini S, Haywood JR, Sandberg K: Sex differences in renal injury and nitric oxide production in renal wrap hypertension. Am J Physiol Heart Circ Physiol 288:H43–H47, 2005[Abstract/Free Full Text]
  26. Forbes JM, Hewitson TD, Becker GJ, Jones CL: Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat. Kidney Int 57:2375–2385, 2000[Medline]
  27. Coughlan MT, Thallas-Bonke V, Pete J, Long DM, Gasser A, Tong DC, Arnstein M, Thorpe SR, Cooper ME, Forbes JM: Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy? Endocrinology 148:886–895, 2007[Abstract/Free Full Text]
  28. Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R, Ping P: Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection. Circ Res 90:390–397, 2002[Abstract/Free Full Text]
  29. Asaba K, Tojo A, Onozato ML, Goto A, Quinn MT, Fujita T, Wilcox CS: Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 67:1890–1898, 2005[Medline]
  30. Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, Abboud HE: Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney. J Biol Chem 280:39616–39626, 2005[Abstract/Free Full Text]
  31. Heitzer T, Wenzel U, Hink U, Krollner D, Skatchkov M, Stahl RA, MacHarzina R, Brasen JH, Meinertz T, Munzel T: Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: evidence for an involvement of protein kinase C. Kidney Int 55:252–260, 1999[Medline]
  32. Bastian NR, Hibbs JB Jr.: Assembly and regulation of NADPH oxidase and nitric oxide synthase. Curr Opin Immunol 6:131–139, 1994[Medline]
  33. Osicka TM, Yu Y, Lee V, Panagiotopoulos S, Kemp BE, Jerums G: Aminoguanidine and ramipril prevent diabetes-induced increases in protein kinase C activity in glomeruli, retina and mesenteric artery. Clin Sci (Lond) 100:249–257, 2001[Medline]
  34. Osicka TM, Yu Y, Panagiotopoulos S, Clavant SP, Kiriazis Z, Pike RN, Pratt LM, Russo LM, Kemp BE, Comper WD, Jerums G: Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C. Diabetes 49:87–93, 2000[Abstract]
  35. Sheetz MJ, King GL: Molecular understanding of hyperglycemia's adverse effects for diabetic complications. JAMA 288:2579–2588, 2002[Abstract/Free Full Text]
  36. Meier M, Park JK, Boehne M, Elger M, Leitges M, Haller H, Menne J: Knockout of protein kinase C {alpha} protects against the development of albuminuria but not renal hypertrophy (Abstract). Diabetes 52:A50, 2003
  37. Wu WS, Tsai RK, Chang CH, Wang S, Wu JR, Chang YX: Reactive oxygen species mediated sustained activation of protein kinase C alpha and extracellular signal-regulated kinase for migration of human hepatoma cell Hepg2. Mol Cancer Res 4:747–758, 2006[Abstract/Free Full Text]
  38. de Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W, Lameire NH: Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. J Am Soc Nephrol 12:993–1000, 2001[Abstract/Free Full Text]
  39. Sung SH, Ziyadeh FN, Wang A, Pyagay PE, Kanwar YS, Chen S: Blockade of vascular endothelial growth factor signaling ameliorates diabetic albuminuria in mice. J Am Soc Nephrol 17:3093–3104, 2006[Abstract/Free Full Text]
  40. Lee BH, Park RW, Choi JY, Ryoo HM, Sohn KY, Kim IS: Stimulation of fibronectin synthesis through the protein kinase C signalling pathway in normal and transformed human lung fibroblasts. Biochem Mol Biol Int 39:895–904, 1996[Medline]
  41. Twigg SM, Joly AH, Chen MM, Tsubaki J, Kim HS, Hwa V, Oh Y, Rosenfeld RG: Connective tissue growth factor/IGF-binding protein-related protein-2 is a mediator in the induction of fibronectin by advanced glycosylation end-products in human dermal fibroblasts. Endocrinology 143:1260–1269, 2002[Abstract/Free Full Text]
  42. Pugliese G, Pricci F, Pugliese F, Mene P, Lenti L, Andreani D, Galli G, Casini A, Bianchi S, Rotella CM, et al.: Mechanisms of glucose-enhanced extracellular matrix accumulation in rat glomerular mesangial cells. Diabetes 43:478–490, 1994[Abstract]
  43. Whiteside CI, Dlugosz JA: Mesangial cell protein kinase C isozyme activation in the diabetic milieu. Am J Physiol Renal Physiol 282:F975–F980, 2002[Abstract/Free Full Text]
  44. Lee HB, Yu MR, Yang Y, Jiang Z, Ha H: Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 14 (Suppl. 3):S241–S245, 2003
  45. Trachtman H, Futterweit S, Pine E, Mann J, Valderrama E: Chronic diabetic nephropathy: role of inducible nitric oxide synthase. Pediatr Nephrol 17:20–29, 2002[Medline]
  46. Xia L, Wang H, Goldberg HJ, Munk S, Fantus IG, Whiteside CI: Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression. Am J Physiol Renal Physiol 290:F345–F356, 2006[Abstract/Free Full Text]
  47. Kelly DJ, Gilbert RE, Cox AJ, Soulis T, Jerums G, Cooper ME: Aminoguanidine ameliorates overexpression of prosclerotic growth factors and collagen deposition in experimental diabetic nephropathy. J Am Soc Nephrol 12:2098–2107, 2001[Abstract/Free Full Text]
  48. Ha H, Lee HB: Oxidative stress in diabetic nephropathy: basic and clinical information. Curr Diab Rep 1:282–287, 2001[Medline]
  49. Rask-Madsen C, King GL: Mechanisms of disease: endothelial dysfunction in insulin resistance and diabetes. Nat Clin Pract Endocrinol Metab 3:46–56, 2007[Medline]

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Comment on: Thallas-Bonke et al. (2008) Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product-Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-{alpha}-Dependent Pathway: Diabetes 57:460-469, 2008
Diabetes, June 1, 2008; 57(6): e13 - e13.
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Response to Comment on: Thallas-Bonke et al. (2008) Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product-Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-{alpha}-Dependent Pathway: Diabetes 57:460-469, 2008
Diabetes, June 1, 2008; 57(6): e14 - e14.
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