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• W2012258975 abstract "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. 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) 4The abbreviations used are:DNdiabetic nephropathyROSreactive oxygen speciesNox4NAD(P)H oxidase 4MCmesangial cellASphosphorothioated antisense oligonucleotidesNGnormal glucoseHGhigh glucoseDCF2′,7′-dichlorodihydrofluoresceinAng IIangiotensin IIERKextracellular signal-regulated kinasePKBprotein kinase BSTZstreptozotocinRLUrelative light unitsPBSphosphate-buffered salineBSAbovine serum albumin (1Bilous R.W. Mauer S.M. Sutherland D.E. Steffes M.W. Diabetes. 1989; 38: 1142-1147Crossref PubMed Scopus (0) Google Scholar, 2Ziyadeh F.N. Hoffman B.B. Han D.C. Iglesias-De La Cruz M.C. Hong S.W. Isono M. Chen S. McGowan T.A. Sharma K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8015-8020Crossref PubMed Scopus (806) Google Scholar, 3Wolf G. Ziyadeh F.N. Kidney Int. 1999; 56: 393-405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 4Ziyadeh F.N. Am. J. Kidney Dis. 1993; 22: 736-744Abstract Full Text PDF PubMed Scopus (231) Google Scholar). Whole kidneys, glomeruli, and tubules undergo hypertrophy by increase in cell size and accumulation of extracellular matrix (3Wolf G. Ziyadeh F.N. Kidney Int. 1999; 56: 393-405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 4Ziyadeh F.N. Am. J. Kidney Dis. 1993; 22: 736-744Abstract Full Text PDF PubMed Scopus (231) Google Scholar). Hypertrophy of the glomerular and tubular compartments precedes the development of irreversible renal changes in diabetes including glomerulosclerosis and tubulointerstitial fibrosis (3Wolf G. Ziyadeh F.N. Kidney Int. 1999; 56: 393-405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 5Phillips A.O. Curr. Diab. Rep. 2003; 3: 491-496Crossref PubMed Scopus (63) Google Scholar). Data from animal models as well as cultured renal cells indicate that hyperglycemia and high glucose induce hypertrophy and extracellular matrix expansion (3Wolf G. Ziyadeh F.N. Kidney Int. 1999; 56: 393-405Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 6Abboud H.E. Kidney Int. 1997; 60: S3-S6Google Scholar, 7Wolf G. Curr. Diab. Rep. 2003; 3: 485-490Crossref PubMed Scopus (37) Google Scholar). diabetic nephropathy reactive oxygen species NAD(P)H oxidase 4 mesangial cell phosphorothioated antisense oligonucleotides normal glucose high glucose 2′,7′-dichlorodihydrofluorescein angiotensin II extracellular signal-regulated kinase protein kinase B streptozotocin relative light units phosphate-buffered saline bovine serum albumin Oxidative stress has emerged as a critical pathogenic factor in the development of DN (8Baynes J.W. Diabetes. 1991; 40: 405-412Crossref PubMed Scopus (0) Google Scholar, 9Hinokio Y. Suzuki S. Hirai M. Chiba M. Hirai A. Toyota T. Diabetologia. 1999; 42: 995-998Crossref PubMed Scopus (178) Google Scholar, 10Sano T. Umeda F. Hashimoto T. Nawata H. Utsumi H. Diabetologia. 1998; 41: 1355-1360Crossref PubMed Scopus (168) Google Scholar, 11Schnackenberg C.G. Curr. Opin. Pharmacol. 2002; 2: 121-125Crossref PubMed Scopus (101) Google Scholar). Diabetes is accompanied by increased generation of reactive oxygen species (ROS) in tissues including the kidney (12Ha H. Kim C. Son Y. Chung M.H. Kim K.H. Free Radic. Biol. Med. 1994; 16: 271-274Crossref PubMed Scopus (108) Google Scholar, 13Onozato M.L. Tojo A. Goto A. Fujita T. Wilcox C.S. Kidney Int. 2002; 61: 186-194Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 14Koya D. Hayashi K. Kitada M. Kashiwagi A. Kikkawa R. Haneda M. J. Am. Soc. Nephrol. 2003; 14: 250-253Crossref PubMed Google Scholar, 15Lee H.B. Yu M.R. Yang Y. Jiang Z. Ha H. J. Am. Soc. Nephrol. 2003; 14: 241-245Crossref PubMed Google Scholar). However, the results of treatment with antioxidants have been inconclusive (16Kuroki T. Isshiki K. King G.L. J. Am. Soc. Nephrol. 2003; 14: 216-220Crossref PubMed Google Scholar). 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) (17Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 18Cui X.L. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3771-3776Crossref PubMed Scopus (165) Google Scholar, 19Jones S.A. Hancock J.T. Jones O.T. Neubauer A. Topley N. J. Am. Soc. Nephrol. 1995; 5: 1483-1491PubMed Google Scholar). Under physiologic conditions, NAD(P)H oxidases have a very low constitutive activity that can be up-regulated in response to various stimuli (15Lee H.B. Yu M.R. Yang Y. Jiang Z. Ha H. J. Am. Soc. Nephrol. 2003; 14: 241-245Crossref PubMed Google Scholar, 17Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 20Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar, 21Li J.M. Shah A.M. J. Am. Soc. Nephrol. 2003; 14: 221-226Crossref PubMed Google Scholar, 22Inoguchi T. Sonta T. Tsubouchi H. Etoh T. Kakimoto M. Sonoda N. Sato N. Sekiguchi N. Kobayashi K. Sumimoto H. Utsumi H. Nawata H. J. Am. Soc. Nephrol. 2003; 14: 227-232Crossref PubMed Google Scholar). For instance, it has been reported that enhanced NAD(P)H oxidase activity is associated with oxidative damage to DNA in diabetic glomeruli (23Kitada M. Koya D. Sugimoto T. Isono M. Araki S. Kashiwagi A. Haneda M. Diabetes. 2003; 52: 2603-2614Crossref PubMed Scopus (194) Google Scholar, 24Etoh T. Inoguchi T. Kakimoto M. Sonoda N. Kobayashi K. Kuroda J. Sumimoto H. Nawata H. Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (226) Google Scholar). These NAD(P)H oxidases are isoforms of the neutrophil oxidase, in which the catalytic subunits, termed Nox proteins, correspond to homologues of gp91phox (or Nox2), the catalytic moiety found in phagocytes (17Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 20Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar). In this family, Nox4, which appears to share the same overall structure with gp91phox/Nox2, is abundant in the vascular system, kidney cortex, and MCs (17Geiszt M. Leto T.L. J. Biol. Chem. 2004; 279: 51715-51718Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 20Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar, 25Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 26Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (710) Google Scholar, 27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar). 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 (20Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar, 22Inoguchi T. Sonta T. Tsubouchi H. Etoh T. Kakimoto M. Sonoda N. Sato N. Sekiguchi N. Kobayashi K. Sumimoto H. Utsumi H. Nawata H. J. Am. Soc. Nephrol. 2003; 14: 227-232Crossref PubMed Google Scholar, 24Etoh T. Inoguchi T. Kakimoto M. Sonoda N. Kobayashi K. Kuroda J. Sumimoto H. Nawata H. Diabetologia. 2003; 46: 1428-1437Crossref PubMed Scopus (226) Google Scholar). We have reported previously that Nox4-derived ROS mediate angiotensin II (Ang II)-induced signaling and protein synthesis in mesangial cells (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar, 28Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (108) Google Scholar), 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. 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 (29Abboud H.E. Ou S.L. Velosa J.A. Shah S.V. Dousa T.P. J. Clin. Investig. 1982; 69: 327-336Crossref PubMed Scopus (23) Google Scholar), 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 (5′-AGCTCCTCCAGGACAGCGCC-3′) (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar, 28Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (108) Google Scholar). 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). Rat glomerular MCs were isolated and characterized as described (30Choudhury G.G. Karamitsos C. Hernandez J. Gentilini A. Bardgette J. Abboud H.E. Am. J. Physiol. 1997; 273: F931-F938PubMed Google Scholar). 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 (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar, 28Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (108) Google Scholar). 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 μl, respectively, of lysis buffer (20 mm KH2PO4, pH 7.0, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 0.5 μg/ml leupeptin) by using a Dounce homogenizer (100 strokes on ice). Homogenates were subjected to a low speed centrifugation at 800 × g, 4 °C, for 10 min to remove the unbroken cells and debris, and aliquots were used immediately. To start the assay, 100 μl of homogenates were added to 900 μl of 50 mm phosphate buffer, pH 7.0, containing 1 mm EGTA, 150 mm sucrose, 5 μm lucigenin, and 100 μm 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 (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar). Briefly, MCs grown in serum-free 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 × 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 NADPH-dependent superoxide generation as above. Measurement of superoxide anion released by isolated glomeruli was performed by detection of superoxide dismutase-inhibitable ferricytochrome c reduction (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar, 31Chen H.C. Guh J.Y. Shin S.J. Tsai J.H. Lai Y.H. J. Lab. Clin. Med. 2000; 135: 309-315Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Isolated glomeruli were incubated in Hanks' balanced salt solution without phenol red containing 80 μm cytochrome c with or without superoxide dismutase (50 μg/ml) for 6 h at 37 °C. At the end of the incubation, glomeruli were centrifuged for 2 min at 10,000 × 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 ΔE550 = 21.0 × 103m-1 cm-1. The reduction of cytochrome c that was inhibitable by pretreatment with superoxide dismutase represents superoxide release. The peroxide-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously (27Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. 2003; 285: F219-F229Crossref PubMed Scopus (242) Google Scholar, 32Gorin Y. Kim N.H. Feliers D. Bhandari B. Choudhury G.G. Abboud H.E. FASEB J. 2001; 15: 1909-1920Crossref PubMed Scopus (96) Google Scholar). This compound is converted by intracellular esterases to 2′,7′-dichlorodihydrofluorescein, which is then oxidized by hydrogen peroxide to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF). Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with ×40 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. In Vivo Experiments—Homogenates from renal cortex were prepared in 500 μl of radioimmune precipitation assay buffer (20 mmol/liter Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1% Nonidet P-40) using a Dounce homogenizer. Homogenates were incubated for 1 h at 4 °C and centrifuged at 10,000 × g for 30 min at 4 °C. Isolated glomeruli were suspended in radioimmune precipitation assay buffer and incubated for 1 h at 4 °C. After centrifugation at 10,000 × 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 serum-free Dulbecco's modified Eagle's medium containing 5 mmd-glucose, 25 mmd-glucose, or 5 mmd-glucose + 20 mml-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 × g for 30 min at 4 °C, and protein was determined in the cleared supernatant using the Bio-Rad method. For immunoblotting, proteins were separated using SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% low fat milk in Tris-buffered saline and then incubated with a mouse polyclonal Nox4 antibody directed against recombinant glutathione S-transferase-mouse Nox4-(299-515) (dilution 1:1,000), a rabbit polyclonal anti-gp91phox (Upstate Biotechnology Inc.) (1:1,000), a rabbit polyclonal anti-fibronectin antibody (Sigma) (1:2,500), or a mouse monoclonal anti-β-actin (1:4,000) and a rabbit anti-phospho-Akt (Ser473) antibody, a rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) antibody, or a rabbit polyclonal anti-Akt1/PKBα (Cell Signaling Technology Inc.) (1:1,000). The appropriate horseradish peroxidase-conjugated secondary antibodies were added, and bands were visualized by enhanced chemiluminescence. Densitometric analysis was performed using the NIH Image software. Light microscopy of hematoxylin and eosin-stained sections from the different treatment groups was used for morphometric studies. The surface area (μm2) 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. 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 μm 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. Six-μm-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 μg/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 Crystal Mount (Dako) and allowed to dry before viewing with fluorescence microscopy. α-Smooth muscle actin was used as a marker for MCs within the glomerulus. Results are expressed as mean ± S.E. Statistical significance was assessed by Student's unpaired t test. Significance was determined as probability (p) less than 0.05. 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 non-diabetic control animals. In contrast, total kidney weight in AS Nox4-treated diabetic rats was significantly reduced compared with that observed for the diabetic or sense Nox4-treated diabetic groups (TABLE ONE).TABLE ONEGlucose level, body weight, kidney weight, and kidney weight to body weight ratio after 2 weeks of diabetesGroupnBlood glucoseBody weightKidney weightKidney weight/body weightmg/dlggg/kgControl10194.9 ± 18.9302.536 ± 5.01.049 ± 0.043.771 ± 0.12Diabetes10386.5 ± 24.2238.5 ± 11.51.273 ± 0.05ap < 0.05 versus control rats.5.526 ± 0.25bp < 0.01 versus control rats.Diabetes + sense Nox410398.2 ± 19.6230.083 ± 11.21.248 ± 0.05ap < 0.05 versus control rats.5.486 ± 0.24bp < 0.01 versus control rats.Diabetes + AS Nox410398.7 ± 40.6223.462 ± 14.61.114 ± 0.04cp < 0.05 versus diabetes.4.476 ± 0.22dp < 0.01 versus diabetes.a p < 0.05 versus control rats.b p < 0.01 versus control rats.c p < 0.05 versus diabetes.d p < 0.01 versus diabetes. Open table in a new tab 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 Nox4-(299-515) showed that a predominant 70-kDa band corresponding to Nox4 was increased in diabetic kidney cortex compared with that in control non-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, gp91phox/Nox2. The levels of gp91phox/Nox2 were also increased in diabetic animals. More importantly, administration of AS Nox4 had no effect on gp91phox/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 α-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 lucigenin-enhanced 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 μg/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 diabetes-induced 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 ± 3.9 in control rats, 433.5 mg/dl ± 25.8 in diabetic rats, and 103.7 mg/dl ± 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 sig" @default.
• W2012258975 created "2016-06-24" @default.
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• W2012258975 date "2005-11-01" @default.
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• W2012258975 title "Nox4 NAD(P)H Oxidase Mediates Hypertrophy and Fibronectin Expression in the Diabetic Kidney" @default.
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