Portal de Periódicos da CAPES

Induction of Glia Maturation Factor-β in Proximal Tubular Cells Leads to Vulnerability to Oxidative Injury through the p38 Pathway and Changes in Antioxidant Enzyme Activities

2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês

10.1074/jbc.m301552200

1083-351X

Jun-Ya Kaimori, Masaru Takenaka, Hideaki Nakajima, Takayuki Hamano, Masaru Horio, Takeshi Sugaya, Takahito Ito, Masatsugu Hori, Kousaku Okubo, Enyu Imai,

Alzheimer's disease research and treatments

Proteinuria is an independent risk factor for progression of renal diseases. Glia maturation factor-β (GMF-β), a 17-kDa brain-specific protein originally purified as a neurotrophic factor from brain, was induced in renal proximal tubular (PT) cells by proteinuria. To examine the role of GMF-β in PT cells, we constructed PT cell lines continuously expressing GMF-β. The PT cells overexpressing GMF-β acquired susceptibility to cell death upon stimulation with tumor necrosis factor-α and angiotensin II, both of which are reported to cause oxidative stress. GMF-β overexpression also promoted oxidative insults by H2O2, leading to the reorganization of F-actin as well as apoptosis in non-brain cells (not only PT cells, but also NIH 3T3 cells). The measurement of intracellular reactive oxygen species in the GMF-β-overexpressing cells showed a sustained increase in H2O2 in response to tumor necrosis factor-α, angiotensin II, and H2O2 stimuli. The sustained increase in H2O2 was caused by an increase in the activity of the H2O2-producing enzyme copper/zinc-superoxide dismutase, a decrease in the activities of the H2O2-reducing enzymes catalase and glutathione peroxidase, and a depletion of the content of the cellular glutathione peroxidase substrate GSH. The p38 pathway was significantly involved in the sustained oxidative stress to the cells. Taken together, the alteration of the antioxidant enzyme activities, in particular the peroxide-scavenging deficit, underlies the susceptibility to cell death in GMF-β-overexpressing cells. In conclusion, we suggest that the proteinuria induction of GMF-β in renal PT cells may play a critical role in the progression of renal diseases by enhancing oxidative injuries. Proteinuria is an independent risk factor for progression of renal diseases. Glia maturation factor-β (GMF-β), a 17-kDa brain-specific protein originally purified as a neurotrophic factor from brain, was induced in renal proximal tubular (PT) cells by proteinuria. To examine the role of GMF-β in PT cells, we constructed PT cell lines continuously expressing GMF-β. The PT cells overexpressing GMF-β acquired susceptibility to cell death upon stimulation with tumor necrosis factor-α and angiotensin II, both of which are reported to cause oxidative stress. GMF-β overexpression also promoted oxidative insults by H2O2, leading to the reorganization of F-actin as well as apoptosis in non-brain cells (not only PT cells, but also NIH 3T3 cells). The measurement of intracellular reactive oxygen species in the GMF-β-overexpressing cells showed a sustained increase in H2O2 in response to tumor necrosis factor-α, angiotensin II, and H2O2 stimuli. The sustained increase in H2O2 was caused by an increase in the activity of the H2O2-producing enzyme copper/zinc-superoxide dismutase, a decrease in the activities of the H2O2-reducing enzymes catalase and glutathione peroxidase, and a depletion of the content of the cellular glutathione peroxidase substrate GSH. The p38 pathway was significantly involved in the sustained oxidative stress to the cells. Taken together, the alteration of the antioxidant enzyme activities, in particular the peroxide-scavenging deficit, underlies the susceptibility to cell death in GMF-β-overexpressing cells. In conclusion, we suggest that the proteinuria induction of GMF-β in renal PT cells may play a critical role in the progression of renal diseases by enhancing oxidative injuries. In chronic nephropathies, proteinuria is reportedly one of the best predictors, independent of mean arterial blood pressure, for disease progression toward end-stage renal failure (1Ruggenenti P. Perna A. Mosconi L. Matalone M. Pisoni R. Gaspari F. Remuzzi G. Kidney Int. Suppl. 1997; 63: S54-S57PubMed Google Scholar, 2Peterson J.C. Adler S. Burkart J.M. Greene T. Hebert L.A. Hunsicker L.G. King A.J. Klahr S. Massry S.G. Seifter J.L. Ann. Intern. Med. 1995; 123: 754-762Crossref PubMed Scopus (1240) Google Scholar). Microalbuminuria, which features a small quantity of albumin only (30–300 mg/24 h), is known as an important early sign of diabetic nephropathy (3Mogensen C.E. N. Engl. J. Med. 1984; 310: 356-360Crossref PubMed Scopus (1764) Google Scholar, 4Viberti G.C. Hill R.D. Jarrett R.J. Argyropoulos A. Mahmud U. Keen H. Lancet. 1982; 1: 1430-1432Abstract PubMed Scopus (1466) Google Scholar) and of progressive renal function loss in a non-diabetic population (5Pinto-Sietsma S.J. Janssen W.M. Hillege H.L. Navis G. De Zeeuw D. De Jong P.E. J. Am. Soc. Nephrol. 2000; 11: 1882-1888PubMed Google Scholar). In experimental models, proteinuria caused tubular insults accompanying infiltration of macrophages and T lymphocytes into the kidney (6Eddy A.A. Am. J. Pathol. 1989; 135: 719-733PubMed Google Scholar). Interstitial inflammation can trigger fibroblast proliferation and accumulation of extracellular matrix proteins, which may facilitate tubulointerstitial fibrosis, which is a hallmark of progression of renal disease. In cultured proximal tubular (PT) 1The abbreviations used are: PT, proximal tubular; GMF-β, glia maturation factor-β; TNF-α, tumor necrosis factor-α; BSO, buthionine sulfoximine; PBS, phosphate-buffered saline; FCS, fetal calf serum; CuZn-SOD, copper/zinc-superoxide dismutase; Mn-SOD, manganesesuperoxide dismutase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; TRITC, tetramethylrhodamine isothiocyanate; ROS, reactive oxygen species; DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; NBT, nitro blue tetrazolium; GPX, glutathione peroxidase; ANOVA, analysis of variance. cells activated by administration of albumin, a number of genes encoding vasoactive and inflammatory molecules, which have potentially toxic effects on the kidney, were transactivated (7Zoja C. Benigni A. Remuzzi G. Exp. Nephrol. 1999; 7: 420-428Crossref PubMed Scopus (60) Google Scholar). These results strongly suggest that altering the disposition of PT cells by proteinuria must be involved in the process of renal damage. However, the mechanisms by which proteinuria accelerates renal disease progression remain largely unknown. We recently found that the brain-specific glia maturation factor-β (GMF-β) gene is induced in PT cells by proteinuria by comparison of the gene expression profiles (8Okubo K. Hori N. Matoba R. Niiyama T. Matsubara K. DNA Seq. 1991; 2: 137-144Crossref PubMed Scopus (76) Google Scholar, 9Okubo K. Hori N. Matoba R. Niiyama T. Fukushima A. Kojima Y. Matsubara K. Nat. Genet. 1992; 2: 173-179Crossref PubMed Scopus (475) Google Scholar) 2Available at bodymap.ims.u-tokyo.ac.jp/. of normal and proteinuria disease models (10Takenaka M. Imai E. Kaneko T. Ito T. Moriyama T. Yamauchi A. Hori M. Kawamoto S. Okubo K. Kidney Int. 1998; 53: 562-572Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 11Nakajima H. Takenaka M. Kaimori J.-y. Nagasawa Y. Kosugi A. Kawamoto S. Imai E. Hori M. Okubo K. Kidney Int. 2002; 61: 1577-1587Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). GMF-β is a 17-kDa brain-specific protein that was isolated from bovine brain homogenate as a substance inducing the maturation of normal neurons as well as glial cells (12Lim R. Hicklin D.J. Ryken T.C. Miller J.F. Brain Res. 1987; 430: 49-57Crossref PubMed Scopus (29) Google Scholar, 13Lim R. Hicklin D.J. Miller J.F. Williams T.H. Crabtree J.B. Brain Res. 1987; 430: 93-100Crossref PubMed Scopus (21) Google Scholar); and at first, it was considered to be a neurotrophic factor. However, later intensive researches provided cumulative evidence that GMF-β is involved in cell signal transduction. This evidence comprised the following findings. 1) The GMF-β protein lacks a leader sequence and is not secreted by cells (14Kaplan R. Zaheer A. Jaye M. Lim R. J. Neurochem. 1991; 57: 483-490Crossref PubMed Scopus (84) Google Scholar). 2) It contains consensus phosphorylation sites and is phosphorylated by protein kinase C, protein kinase A, casein kinase II, and ribosomal S6 kinase in in vitro studies (15Lim R. Zaheer A. Biochem. Biophys. Res. Commun. 1995; 211: 928-934Crossref PubMed Scopus (30) Google Scholar, 16Zaheer A. Lim R. J. Biol. Chem. 1997; 272: 5183-5186Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 3) GMF-β inhibits the extracellular signal-regulated kinase-1/2 and enhances p38 activity (17Lim R. Zaheer A. J. Biol. Chem. 1996; 271: 22953-22956Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 4) Overexpressed GMF-β in primary astrocytes causes secretion of neurotrophic factors such as brain-derived neurotrophic factor and nerve growth factor through activation of the p38 pathway (18Zaheer A. Yorek M.A. Lim R. Neurochem. Res. 2001; 26: 1293-1299Crossref PubMed Scopus (67) Google Scholar). The amino acid sequence of GMF-β is highly conserved among many species (19Zaheer A. Fink B.D. Lim R. J. Neurochem. 1993; 60: 914-920Crossref PubMed Scopus (66) Google Scholar), suggesting that it plays basic roles across many species. The expression of GMF-β is largely limited to the brain (19Zaheer A. Fink B.D. Lim R. J. Neurochem. 1993; 60: 914-920Crossref PubMed Scopus (66) Google Scholar), especially the glial cells and some neurons (20Wang B.R. Zaheer A. Lim R. Brain Res. 1992; 591: 1-7Crossref PubMed Scopus (30) Google Scholar). Schwann cells of the distal segment of the transected nerve express GMF-β, and this induction of GMF-β coincides with the temporal expression of nerve growth factor receptors in the cells (21Bosch E.P. Zhong W. Lim R. J. Neurosci. 1989; 9: 3690-3698Crossref PubMed Google Scholar). These results suggest that GMF-β may play a protective role in the brain. However, its precise function in neurons and glial cells is still largely a matter of speculation. It was thus of interest to determine what impact could be served by the induction of the brain-specific protein GMF-β in PT cells by proteinuria. In this study, we demonstrate that proteinuria induced GMF-β in PT cells in a time-dependent manner. Overexpression of GMF-β in a mouse PT cell line and NIH 3T3 cell lines resulted in susceptibility to cell death upon stimulation with tumor necrosis factor-α (TNF-α), angiotensin II, and other oxidative stress-inducing agents (buthionine sulfoximine (BSO) and H2O2). Further studies clarified that GMF-β overexpression caused enhancement of oxidative injury, leading to F-actin reorganization and apoptosis under oxidative stress. We demonstrate that GMF-β-overexpressing cells, as a mechanism of the vulnerability to oxidative stress, caused a prolonged increase in H2O2 through changes in several antioxidant enzyme activities and that p38 was significantly involved in this process. These results suggest that the induction of the brain-specific GMF-β gene in the kidney may play a key role in renal disease progression caused by proteinuria. Protein-overloaded Proteinuria Murine Model—Five-week-old C57BL6 male and female mice weighing ∼20 g were intraperitoneally given bovine serum albumin (10 mg/g of weight; Sigma) dissolved in saline for 5 days during a 1-week period. The final dose of 10 mg/g of weight was reached by incremental increases in the dose over the first week, beginning with 2.5 mg/g of weight. Control mice were treated with saline (7Zoja C. Benigni A. Remuzzi G. Exp. Nephrol. 1999; 7: 420-428Crossref PubMed Scopus (60) Google Scholar, 11Nakajima H. Takenaka M. Kaimori J.-y. Nagasawa Y. Kosugi A. Kawamoto S. Imai E. Hori M. Okubo K. Kidney Int. 2002; 61: 1577-1587Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The load of bovine serum albumin was continued to 4 weeks. At 1, 2, 3, and 4 weeks, 24 h after loading of bovine serum albumin, kidneys were removed for investigations. Tissue Preparation and Laser Capture Microdissection—The protein-overloaded proteinuria model mice were separated into three random groups. After 1, 2, 3, or 4 weeks of loading of bovine serum albumin, kidneys from the first group (n = three each) were removed after perfusion with phosphate-buffered saline (PBS) for Northern blot analysis, and those from the second group (n = one each) after perfusion first with PBS and then with 4% paraformaldehyde. They were made into specimens with the paraffin sectioning method after paraformaldehyde fixation and used for in situ hybridization. Kidneys from the third group (n = three each) were removed after perfusion first with PBS and then with 99.5% ethanol. Kidney tissue sections were prepared and subjected to laser microdissection using an LM200 Image Archiving workstation (Arcturus Engineering, Mountain View, CA) along with real-time PCR as described (25Nagasawa Y. Takenaka M. Matsuoka Y. Imai E. Hori M. Kidney Int. 2000; 57: 717-723Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 26Kaimori J.-y. Takenaka M. Nagasawa Y. Nakajima H. Izumi M. Akagi Y. Imai E. Hori M. Am. J. Kidney Dis. 2002; 39: 948-957Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Total RNA was extracted from samples attached to LCM transfer film using TRIzol (Invitrogen) and reverse-transcribed with SuperScript™ II RNase H reverse transcriptase (Invitrogen). Quantitation of GMF-β mRNA/rRNA was performed with this real-time PCR system according to the manufacturer's instructions. The GMF-β TaqMan probe was 5′-TGGTTTCAGTCTCTGCTAGTTCATACCGCA-3′. The GMF-β forward primer sequence was 5′-GAGGCTTGAAACATTGGTGGTT-3′, and its reverse primer sequence was 5′-CAAGCACCATGCTTACCAAAAG-3′. The rRNA TaqMan probe and the forward and reverse primers were obtained from TaqMan rRNA control reagents (Applied Biosystems, Foster City, CA). Northern Blot Analysis—Total RNA from mouse kidney was extracted with TRIzol according to the manufacturer's instructions. Ten micrograms of the RNAs were fractionated on formaldehyde-agarose gels, transferred onto nylon membranes (Hybond-N+, Amersham Biosciences), and subjected to Northern analyses. For the probes, we used the mouse GMF-β sequences (bp 3080–4130) obtained from the GenBank™/EBI Data Bank (accession number AF297220). Glyceraldehyde-3-phosphate dehydrogenase cDNA was used as an internal control. In Situ Hybridization—GMF-β was subjected to in situ hybridization with the DNA nucleic acid detection kit (Roche Applied Science) according to the manufacturer's instructions. The GMF-β sense and antisense cRNA probes were prepared by PCReaction. The primers for PCR were 5′-GTCGCAGTAGAGTGGAGTGTGTTG-3′ and 5′-CAGGTCAGGGCCATTCACTCTATG-3′. The PCR product was then subcloned into pST-Blue-1, and the sequence was confirmed to be identical to that of mouse GMF-β (data not shown). The clone was digested by XhoI to produce the template of the antisense probe and by BamHI to produce that of the sense probe. The digoxigenin-labeled antisense and sense cRNA probes were generated using 1 μg of template and T7 or SP6 RNA polymerase, respectively, in combination with the digoxigenin RNA labeling mixture (Roche Applied Science). Cell Culture and Transfection—The mouse renal proximal tubular cell line mProx24 (60Yamanouchi, M., Honda, A., Uchida, M., Kimura, K., and Sugaya, T. (October 11, 2000) International Patent WO0073791Google Scholar) and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal calf serum (FCS) (Invitrogen) in 5% CO2 at 37 °C. The mProx24 cells were maintained in 0.1% gelatin-coated culture dishes. The mouse GMF-β coding region was amplified by PCR using forward primer 5′-CGGGATCCCGCTGACGACCGGAAGGAAAATGAGTGAG-3′ and reverse primer 5′-CGGGATCCCGCCAGTACCCAGGAGTGGTCAGAGGAGG-3′, cut with BamHI, and inserted into the BglII site of FLAG-linked pCMV-Taq1 (Stratagene, La Jolla, CA). Permanent transfection of cells with mammalian expression vectors was achieved with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. The transfectants were maintained in Dulbecco's modified Eagle's medium containing 10% FCS and 200 μg/ml Geneticin (G418, Sigma). Immunoblotting—Total cell lysates were separated by SDS-PAGE (12% acrylamide) and then electrotransferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). FLAG-GMF-β was detected using mouse monoclonal anti-FLAG antibody M2 (Sigma) at a 5000:1 dilution. The blots were probed with individual primary antibodies as described above and then incubated with horseradish peroxidase-conjugated antibodies. Rabbit polyclonal antibody was raised against mouse GMF-β (the amino acid sequence of the epitope polypeptide was NH2-YQHDDGRVSYPLC-COOH) and confirmed by immunoblotting to bind specifically the GMF-β protein band. Anti-CuZn-SOD and anti-Mn-SOD antibodies were purchased from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). Proteins were visualized with an enhanced chemiluminescence reagent (Pierce). Cell Viability Assay—Cell viability for mouse PT and NIH 3T3 cells was determined 84 and 48 h, respectively, after treatments by the addition of the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (inner salt; Promega) (27Cory A.H. Owen T.C. Barltrop J.A. Cory J.G. Cancer Commun. 1991; 3: 207-212Crossref PubMed Scopus (1319) Google Scholar). The MTS tetrazolium compound is bioreduced by cells into a colored formation product that is soluble in culture medium. The solution of MTS mixed with the electron-coupling reagent phenazine methosulfate was added directly to the culture wells and incubated for 1 h, after which absorbance at 490 nm was recorded with a 96-well plate reader. Mouse TNF-α, angiotensin II, H2O2, and BSO were purchased from Sigma (Sigma). Lactate Dehydrogenase Release Assay—The results obtained in the MTS assay were confirmed in a lactate dehydrogenase release assay. A cytotoxicity detection kit (Roche Applied Science) was used to quantitate cytotoxicity/cytolysis based on the measurement of lactate dehydrogenase activity released from damaged cells. The lactate dehydrogenase activities in the culture supernatants of mProx24 and NIH 3T3 cells were determined 48 and 24 h, respectively, after treatments according to the manufacturer's instructions. Detection of Apoptotic Cells by Annexin V Flow Cytometer Analysis— Detection of apoptotic cells was performed with an annexin V-enhanced green fluorescent protein apoptosis detection kit (Medical & Biological Laboratories, Nagoya, Japan). The assay takes advantage of the properties of binding of annexin V to membrane phosphatidylserine, which is translocated from the inner face of the plasma membrane to the apoptotic cell surface to make early detection of apoptosis by flow cytometry possible. After the addition of H2O2 following one overnight FCS starvation, mProx24 cells were gently trypsinized and washed once with 10% FCS-containing Dulbecco's modified Eagle's medium before incubation with annexin V-enhanced green fluorescent protein at room temperature for 5 min in the dark. The analysis of annexin V-enhanced green fluorescent protein binding was performed with a FACSCalibur flow cytometry (excitation at 488 nm and emission at 530 nm; BD Biosciences) using a fluorescein isothiocyanate signal detector. Activities of Caspase-3—Caspase-3 activities were detected using a colorimetric assay kit (Promega). Ac-DEVD-p-nitroaniline was used as the substrate for caspase-3. p-Nitroaniline was released from the substrate after cleavage with the DEVDase caspase-3. Free p-nitroaniline produced a yellow color that was monitored with a Emax microplate reader (Molecular Devices, Sunnyvale, CA) at 405 nm. After this treatment, the adherent mProx24 cells, both the stable transfectants and wild-type cells, were scraped off and washed with PBS by centrifugation. The cells were then resuspended in hypotonic cell lysis buffer (5 mm EDTA, 5 mm dithiothreitol, 25 mm HEPES (pH 7.5), 5 mm MgCl2, 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, and 10 μg/ml leupeptin) and lysed by three cycles of freezing and thawing. The cells were finally centrifuged at 16,000 × g for 20 min at 4 °C, and the supernatants were used as samples for caspase assay. The protein contents of the same supernatants were assayed with the Bio-Rad protein assay. Benzyloxycarbonyl-VAD-fluoromethyl ketone (50 μm) was included in the caspase-3 assay kit. Detection of Nucleosomal Ladders in Apoptotic Cells—DNA was extracted using the DNAzol reagent (Invitrogen). To detect nucleosomal ladders for apoptotic cells, the PCR-based amplification kit for DNA ladders (Clontech, Palo Alto, CA) was used according to the manufacturer's instructions. Staining of F-actin and Confocal Fluorescence Microscopy—Before the various treatments, dish-cultured cells were transferred to 0.1% gelatin-coated coverslips. After 24 h, the medium was removed, and the cells were starved in serum-free medium for 12 h, after which they were fixed with 3.7% formaldehyde and permeated with 0.5% Triton X-100 (Sigma) in PBS (pH 7.5). F-actin was detected using 0.165 μm TRITC-labeled phalloidin (Sigma). The cells were examined by confocal microscopy using a Radiance 2100 BLD (Bio-Rad). Determination of Reactive Oxygen Species (ROS) Production in PT Cells—Intracellular ROS generation was assessed using the oxidant-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) (Lambda Fluoreszenztechnologie, Graz, Austria) (28Su B. Mitra S. Gregg H. Flavahan S. Chotani M.A. Clark K.R. Goldschmidt-Clermont P.J. Flavahan N.A. Circ. Res. 2001; 89: 39-46Crossref PubMed Scopus (138) Google Scholar) in wild-type PT cells and those stably overexpressing GMF-β. One of the ROS, intracellular H2O2, could be assayed (29Thannickal V.J. Fanburg B.L. Am. J. Physiol. 2000; 279: L1005-L1028Crossref PubMed Google Scholar). PT cells were treated with 5 μg/ml DCFDA for 30 min at 37 °C in Krebs-Ringer bicarbonate solution (118.3 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 2.5 mm CaCl2, 25.0 mm NaHCO3, and 11.1 mm glucose). After DCFDA incubation, the attached PT cells were cooled and harvested by trypsinization at 4 °C, followed by flow cytometry analysis using a FACSCalibur. To examine the influence of intracellular esterase and probe efflux on the DCFDA assay, the oxidant-insensitive fluorescein diacetate derivative carboxyfluorescein diacetate (Molecular Probes, Inc., Eugene, OR) was used at 10 μm. The assay method using carboxyfluorescein diacetate was the same as that using DCFDA. SOD Activity Assay—SOD activity was measured as described by Sutherland and Learmonth (30Sutherland M.W. Learmonth B.A. Free Radic. Res. 1997; 27: 283-289Crossref PubMed Scopus (223) Google Scholar) using an SOD assay kit (Trevigen, Gaithersburg, MD). This method is based on the inhibition of the reduction of nitro blue tetrazolium (NBT) by SOD. Superoxide ions convert NBT into NBT-diformazan. NBT-diformazan absorbs light at 550 nm. SOD reduces the superoxide ion concentration and thereby lowers the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBT-formazan reflects the amount of SOD activity in a sample. CuZn-SOD was extracted with an ethanol/chloroform method, and its activity was measured. Mn-SOD activity was determined by subtracting CuZn-SOD activity from total SOD activity. Catalase Activity Assay—Catalase activity was measured as described by Zhou et al. (31Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1126) Google Scholar) using the Amplex® Red catalase assay kit (Molecular Probes, Inc.). Cells were lysed and processed in isotonic buffer (10 mm Tris-Cl (pH 7.4), 200 mm mannitol, 50 mm sucrose, and 1 mm EDTA). In the assay, catalase first reacts with H2O2 to produce water and oxygen. Next, the Amplex Red reacts, as calculated with a 1:1 stoichiometry, with any unreacted H2O2 in the presence of horseradish peroxidase to produce the highly fluorescent oxidized product resorufin. Since resorufin has also strong absorption of 563 nm, detection of this absorbance made it possible to determine the quantity of resorufin and therefore catalase activity. Glutathione Peroxidase (GPX) Activity Assay—GPX activity was measured as described by Paglia and Valentine (32Paglia D.E. Valentine W.N. J. Lab. Clin. Med. 1967; 70: 158-169PubMed Google Scholar) using a GPX assay kit (Oxis Research, Portland, OR). This assay is an indirect measure of the activity of cellular GPX. Oxidized glutathione is recycled to its reduced form by glutathione reductase. The oxidation of NADPH resulting in NAD+ is accompanied by a decrease in absorbance at 340 nm so that GPX activity can be monitored. To assay cellular GPX, a cell homogenate is added to a solution containing glutathione, glutathione reductase, and NADPH. The enzyme reaction is started by adding t-butyl hydroperoxide as a substrate, and the absorbance at 340 nm is recorded every 30 s for 3 min. The rate of decrease in the absorbance at 340 nm is directly proportionate to the GPX activity in the sample. Cellular Glutathione Content Assay—Cellular glutathione content was measured using a glutathione content assay kit (GSH-400™, Oxis Research). The method used with this kit is based on a chemical reaction that proceeds in two steps. The first step is the formation of substitution products (thioethers) between 4-chloro-1-methyl-7-trifluromethylquinolinium methylsulfate and all mercaptans that are present in the sample. The second step is a β-elimination reaction under alkaline conditions, which transforms the substitution product (thioether) obtained with glutathione into a thione with a maximal absorbance at 400 nm. Cell pellets are resuspended in an ice-cold metaphosphoric acid working solution, and the cell lysate is produced by homogenizing the cell suspension. Statistical Analyses—Values are expressed as means ± S.D. Except for the fluorescence-activated cell sorter analyses, all values were derived from measurements done in triplicate. Statistical analyses for multiple comparisons were performed with analysis of variance (ANOVA) and post hoc Bonferroni's correction. Unpaired Student's t test was used for the comparison of two groups in the studies with NIH 3T3 cells (Figs. 3, 4, and 8). A p value <0.05 was considered to indicate statistical significance. ANOVA and unpaired Student's t test were performed using a StatView software package (Abacus Concepts Inc., Berkeley, CA).Fig. 4Cell viability of GMF-β-overexpressing and mock-transfected PT and NIH 3T3 cells after challenge with H2O2 and BSO. Each cell line was cultured on 96-well plates for 1 day and treated with 250 μm H2O2 and 500 μm BSO for 84 h for PT cells and for 48 h for NIH 3T3 cells. Cell viability was determined with the MTS assay. Each graph shows the mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001 versus wild-type cells (ANOVA (mProx24 cells) and Student's t test (NIH 3T3 cells)). WT, wild-type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8Antioxidant enzyme activity and GSH content in mouse GMF-β-overexpressing PT and NIH 3T3 cell lines. A, CuZn-SOD immunoblotting (upper) and activity (lower) in GMF-β-overexpressing (GMF) and mock-transfected wild-type (WT) PT (left) and NIH 3T3 (right) cell lines. GMF-β stable transformants showed an increase in CuZn-SOD protein expression and activity. B, Mn-SOD immunoblotting (upper) and activity (lower) in GMF-β-overexpressing and mock-transfected wild-type PT (left) and NIH 3T3 (right) cell lines. Expression of the Mn-SOD protein and its activity were not different in the wild-type cells and GMF-β stable transformants. C, catalase activity in GMF-β-overexpressing and mock-transfected wild-type PT (left) and NIH 3T3 (right) cell lines. Catalase activity decreased in GMF-β-overexpressing cell lines. D, cellular glutathione content in GMF-β-overexpressing and mock-transfected wild-type PT (left) and NIH 3T3 (right) cell lines. Glutathione content was reduced in GMF-β-overexpressing cell lines. E, GPX activity in GMF-β-overexpressing and mock-transfected wild-type PT (left) and NIH 3T3 (right) cell lines. GPX activity in GMF-β-overexpressing cell lines was highly reduced compared with that in wild-type cells. Each graph shows the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus wild-type cells (ANOVA (mProx24 cells) and Student's t test (NIH 3T3 cells)).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Northern Blot Analysis—The gene expression profile of the proximal tubules of the proteinuria mouse model (11Nakajima H. Takenaka M. Kaimori J.-y. Nagasawa Y. Kosugi A. Kawamoto S. Imai E. Hori M. Okubo K. Kidney Int. 2002; 61: 1577-1587Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) confirmed that the expression of GMF-β was up-regulated by proteinuria in 1 week.2, 3Available at www.med.osaka-u.ac.jp/pub/medone/kidney/array/index.html. To examine the gene expression of GMF-β in the diseased mouse model kidney after >1 week of proteinuria, Northern blot analysis was performed using RNA from kidney tissues of control and diseased mouse models. The gene expression of GMF-β was found increase in a time-dependent manner (Fig. 1A), resulting in a significant increase in GMF-β gene expression after 2 weeks of proteinuria. In Situ Hybridization of GMF-β in the Kidney—To examine the localization of GMF-β mRNA expression in the diseased mouse model kidney, in situ hybridization was performed. An increase in GMF-β gene expression was seen mainly in the proximal tubules after 3 weeks of proteinuria (Fig. 1B), whereas no staining was detected in