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• W2147339154 abstract "Diabetic nephropathy affects 30–40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product ϵ(γ-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM ϵ(γ-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated ϵ(γ-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor β1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor β1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated. Diabetic nephropathy affects 30–40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product ϵ(γ-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM ϵ(γ-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated ϵ(γ-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor β1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor β1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated. Diabetic nephropathy (DN) 1The abbreviations used are: DN, diabetic nephropathy; tTg, tissue transglutaminase; Tg, transglutaminase; ECM, extracellular matrix; TGFβ1, transforming growth factor β1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PTCs, proximal tubular epithelial cells; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; TMB, 3,3′,5,5′-tetramethylbenzidine; DOC, sodium deoxycholate; MOPS, 4-morpholinepropanesulfonic acid. accounts for 30–40% of patients requiring renal replacement therapy (1Amos A.F. McCarty D.J. Zimmet P. Diabet. Med. 1997; 14: S1-S85Crossref PubMed Google Scholar, 2Chiarelli F. Trotta D. Verrotti A. Mohn A. Panminerva. Med. 2003; 45: 23-41PubMed Google Scholar, 3Gilbert R.E. Cooper M.E. Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). It is characterized by an early thickening of tubular and glomerular basement membranes due to an excessive accumulation of the extracellular matrix and ultimately leads to progressive scarring and fibrosis of the kidney (1Amos A.F. McCarty D.J. Zimmet P. Diabet. Med. 1997; 14: S1-S85Crossref PubMed Google Scholar, 2Chiarelli F. Trotta D. Verrotti A. Mohn A. Panminerva. Med. 2003; 45: 23-41PubMed Google Scholar, 3Gilbert R.E. Cooper M.E. Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). It also causes a significant increase in the cardiovascular morbidity and mortality of diabetic patients. The incidence of diabetes and diabetic nephropathy continues to rise worldwide. It is predicted that the number of people with diabetes will continue to increase, reaching 221 million by 2010 (1Amos A.F. McCarty D.J. Zimmet P. Diabet. Med. 1997; 14: S1-S85Crossref PubMed Google Scholar). Clinically, DN manifests itself by the onset of continuous microalbuminuria followed by the appearance of persistent proteinuria (2Chiarelli F. Trotta D. Verrotti A. Mohn A. Panminerva. Med. 2003; 45: 23-41PubMed Google Scholar). This is followed by a progressive decline in glomerular filtration rate ultimately leading to end-stage renal failure. Although DN was traditionally considered to be primarily a glomerular disease, it is now widely accepted that the rate of deterioration of kidney function correlates best with the degree of tubulointerstitial fibrosis (3Gilbert R.E. Cooper M.E. Kidney Int. 1999; 56: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Consequently, increased research has focused on the role of the tubular epithelial cells, in particular the proximal tubular epithelial cells, in the initiation of fibrosis. In a recent study using the rat streptozotocin-induced hyperglycemic model of diabetes, we demonstrated elevated levels of the protein cross-linking enzyme tissue transglutaminase (tTg) in the peritubular interstitial space. This change in enzyme distribution was paralleled by an increase in the ϵ(γ-glutamyl)-lysine protein cross-link (4Skill N.J. Griffin M. El Nahas A.M. Sanai T. Haylor J.L. Fisher M. Jamie M.F. Mould N.N. Johnson T.S. Lab. Invest. 2001; 81: 705-716Crossref PubMed Scopus (48) Google Scholar). We have shown similar changes in fibrotic/scarred human kidney biopsy material (5Johnson T.S. El-Koraie A.F. Skill N.J. Baddour N.M. El Nahas A.M. Njloma M. Adam A.G. Griffin M. J. Am. Soc. Nephrol. 2003; 14: 2052-2062Crossref PubMed Scopus (98) Google Scholar) and in the rat remnant kidney model of renal scarring (6Johnson T.S. Griffin M. Thomas G.L. Skill J. Cox A. Yang B. Nicholas B. Birckbichler P.J. Muchaneta-Kubara C. Meguid El Nahas A. J. Clin. Invest. 1997; 99: 2950-2960Crossref PubMed Scopus (122) Google Scholar, 7Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar). In all cases the up-regulation of tTg and its product correlated significantly with increased interstitial fibrosis and scarring. In both animal and human tissues, we demonstrated tubular epithelial cells to be the major source of tTg. Tissue transglutaminase belongs to a group of calcium-dependent mammalian enzymes that have the capacity to irreversibly cross-link proteins through the formation of ϵ(γ-glutamyl)lysine bonds (8Griffin M. Casadio R. Bergamini C. Biochem. J. Rev. 2002; 368: 377-396Crossref PubMed Scopus (0) Google Scholar). In addition to the potential role in kidney scarring, elevated levels of tTg have been associated with a number of other fibrotic diseases, including lung, liver, and heart (9Griffin M. Smith L.L. Wynne J. Br. J. Exp. Pathol. 1979; 60: 653-661PubMed Google Scholar, 10Small K. Feng J.F. Lorenz J. Donnelly E.T. Yu A. Im M.J. Dorn 2nd, G.W. Liggett S.B. J. Biol. Chem. 1999; 274: 21291-21296Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 11Mirza A. Liu S.L. Frizell E. Zhu J. Maddukuri S. Martinez J. Davies P. Schwarting R. Norton P. Zern M.A. Am. J. Physiol. 1997; 272: G281-G288PubMed Google Scholar), where its ability to cross-link ECM is thought to facilitate their increased deposition (12Gross S.R. Balklava Z. Griffin M. J. Invest. Dermatol. 2003; 121: 412-423Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Kleman J.P. Aeschlimann D. Paulsson M. van der Rest M. Biochemistry. 1995; 34: 13768-13775Crossref PubMed Scopus (103) Google Scholar) as well as their increased resistance to the action of matrix metalloproteinases (7Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar). In addition to this proposed direct effect of tTg on matrix accumulation, the enzyme has also been implicated in the matrix storage and subsequent activation of the fibrogenic cytokine, transforming growth factor β1 (TGFβ1), through the cross-linking of the latent TGFβ1-binding protein (LTBP-1) to the ECM (14Nunes I. Gleizes P.E. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (348) Google Scholar) thus facilitating its activation. Given these potential direct and indirect contributions of tTg to the pathogenesis of DN and the epidemiological evidence, which indicates that the complications of diabetes are related to poor glycemic control (2Chiarelli F. Trotta D. Verrotti A. Mohn A. Panminerva. Med. 2003; 45: 23-41PubMed Google Scholar, 15Gaster B. Hirsch I.B. Arch. Intern. Med. 1998; 158: 134-140Crossref PubMed Scopus (182) Google Scholar), it was important to demonstrate a molecular link between elevated glucose levels and changes in the expression and action of tissue transglutaminase. To date, changes in tTg in relation to elevated glucose conditions have only been observed in the pancreatic β cell (16Bungay P.J. Owen R.A. Coutts I.C. Griffin M. Biochem. J. 1986; 235: 269-278Crossref PubMed Scopus (60) Google Scholar). The aim of the current study is to investigate the effects of high glucose levels on tTg expression and activity in the well characterized opossum proximal tubular epithelial cell line (OK cells) (17Gstraunthaler G. Seppi T. Pfaller W. Zhang S.L. Filep J.G. Hohman T.C. Tang S.S. Ingelfinger J.R. Chan J.S. Tam V.K. Clemens T.L. Green J. Cell Physiol. Biochem. 1999; 9: 150-172Crossref PubMed Scopus (77) Google Scholar, 18Tam V.K. Clemens T.L. Green J. Endocrinology. 1998; 139: 3072-3080Crossref PubMed Scopus (9) Google Scholar, 19Zhang S.L. Filep J.G. Hohman T.C. Tang S.S. Ingelfinger J.R. Chan J.S. Tam V.K. Clemens T.L. Green J. Kidney Int. 1999; 55: 454-464Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Initial studies were aimed at examining the regulation of tTg expression in response to elevated glucose concentrations, whereas subsequent studies examined how changes in tTg could affect ECM deposition. We demonstrate that elevated glucose concentrations stimulate de novo synthesis of tTg, which is paralleled by an increase in the expression of the ECM proteins fibronectin, collagen III, and collagen IV, with a subsequent increase in the deposition of total collagen and fibronectin. In the time frame of our experiments (96 h) we observed no changes in the expression or presence of TGFβ1. We demonstrate, for the first time, that the observed increase in the deposition of fibronectin, collagen, and other matrix proteins induced by elevated glucose conditions can be directly linked to their covalent cross-linking via ϵ(γ-glutamyl)lysine bridges. Experimental Protocol and Conditions—Opossum kidney proximal tubular epithelial cells (OK PTCs) (European collection of cell cultures, Cambridge, UK) were cultured in Dulbecco's modified Eagle's medium containing 6 mm d-glucose (glucose-free Dulbecco's modified Eagle's medium (Life Technologies) plus 1.1g/liter d-glucose), 100 units/ml penicillin, 100 μg/ml streptomycin, 20 mm glutamine, and 10% (v/v) fetal calf serum in a humidified atmosphere at 5% (v/v) CO2. For experimentation, 6 mm d-glucose was considered equivalent to normal glycemic conditions. Glucose concentrations between 12 and 36 mm were considered to represent the clinical hyperglycemic environment. To achieve this, media was further supplemented with d-glucose to the required concentration with a parallel flask containing an equivalent concentration of l-glucose to control for osmolarity. All experiments were carried out over a 96-h period with the alteration to glucose at the time of plating. To ensure that glucose differentials were maintained throughout the experimental time period, media was assayed for glucose daily using a glucose analyzer (Analox P-GM7 Micro-Stat) and glucose added to compensate for cell usage. For Tg inhibition studies, two highly specific active site-directed, irreversible inhibitors were used. One, 1,3-dimethyl-2[(oxopropyl)thio]-imidazolium (20Fround K.F. Doshi K. Gaul S. Biochemistry. 1994; 33: 10109-10119Crossref PubMed Scopus (103) Google Scholar, 21.Syntex (March, 1990) U. S. Patent 4,912,120Google Scholar) was termed compound NTU283. The other, a carboxybenzoyl-glutamylglycine analogue, was termed compound NTU281. 2Patent application number GB0314262.7, June 2003. Both compounds were synthesized in-house. The inhibitor NTU283 was added to 36 mm d-glucose culture media at the time of plating to a final concentration of 33, 66, or 99 μm. The inhibitor NTU281 was used in the collagen fibrillogenesis assays at a concentration of 250 μm. Cell Number, Protein, DNA, and Viability Determination—Protein concentrations were determined using protocols based on the Lowry and the bicinchoninic acid assays. DNA was determined using the Burton assay (diphenylamine). Viable cell numbers were measured by the counting of cells using the trypan blue exclusion method. Cell viability/cell leakage was also determined by measuring leakage of lactate dehydrogenase into the cell culture medium (Cytotox 96 viability assay kit, Promega, Southampton, UK). Quantitation of Transglutaminase—Unless specified, cells were removed with 2 mg/ml trypsin-2 mm EDTA then centrifuged at 300 × g for 5 min, and the resultant pellet was washed in PBS. Cells were resuspended in 250 μl of STE buffer (0.32 m sucrose, 5 mm Tris, 1 mm EDTA) containing protease inhibitors phenylmethylsulfonyl fluoride (1 mm), benzamidine (5 mm), and leupeptin (10 μg/ml) and homogenized on ice. For extracellular Tg, the media was collected and cells were removed by centrifugation. The media was freeze-dried and re-suspended in 1/10 volume of STE buffer with protease inhibitors prior to analysis for activity and tTg antigen (12Gross S.R. Balklava Z. Griffin M. J. Invest. Dermatol. 2003; 121: 412-423Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Transglutaminase Activity—Transglutaminase activity was determined by the Ca2+-dependent incorporation of [1,4-14C]putrescine (Amersham Biosciences) into N,N′-dimethylcasein as previously described (6Johnson T.S. Griffin M. Thomas G.L. Skill J. Cox A. Yang B. Nicholas B. Birckbichler P.J. Muchaneta-Kubara C. Meguid El Nahas A. J. Clin. Invest. 1997; 99: 2950-2960Crossref PubMed Scopus (122) Google Scholar). Results were corrected to units/mg of protein (1 unit equals 1 nmol of putrescine incorporated per hour at 37 °C). tTg Western Blotting—Proteins were separated on a 10% (w/v) polyacrylamide gel and then electroblotted onto Hybond ECL (Amersham Biosciences). The membrane was blocked with 3% nonfat dried milk, 0.05% (v/v) Tween 20 in PBS (milk-Tween-PBS) and then immunoprobed with 0.2 μg/ml of a anti-tTg monoclonal antibody (CUB7402, Stratek Scientific, Luton, UK) at 4 °C overnight. Primary antibody binding was revealed with 1.5 μg/ml goat anti-mouse horseradish peroxidase secondary antibody (Dako, UK) for 1 h at room temperature and visualized using ECL chemiluminescence (Amersham Biosciences). Films were quantified by volume densitometry using a Bio-Rad GS-690 imaging densitometer and molecular analyst version 4 software (Bio-Rad). tTg ELISA—Ninety-six-well plates were coated with 100 μl of 5 μg/ml fibronectin overnight at 4 °C and blocked as above. Cell homogenates or concentrated culture media (50 μl) were incubated in triplicate for 2 h at room temperature and then washed with milk-Tween-PBS. Wells were then immunoprobed for tTg with 100 μl of 0.2 μg/ml of anti-tTg monoclonal antibody CUB7402 overnight at 4 °C and revealed with 100 μl of 1.5 μg/ml goat anti-mouse horseradish peroxidase-conjugated antibody for 2 h at room temperature using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. 50 μl of 2.5 m H2SO4 was used to stop the reaction, and the absorbance was read at 450 nm. Immunolocalization of tTg—OK cells were seeded and cultured in 8-well plastic chamber slides (Lab Tech, Scientific Laboratory Supplies, Nottingham, UK), incubated for 96 h under experimental conditions and immunoprobed for tTg, as previously described (22Verderio E. Gaudry C. Gross S. Smith C. Downes S. Griffin M. J. Histochem. Cytochem. 1999; 47: 1417-1432Crossref PubMed Scopus (91) Google Scholar) using 0.6 μg/ml of anti-tTg monoclonal antibody CUB7042 overnight at 4 °C. Binding was revealed using 3 μg/ml goat anti-mouse secondary antibody conjugated to fluorescein and visualized using a Leica TCS-NT confocal laser microscope with excitation at 488 nm and the emissions recorded at 530 nm. Extracellular Matrix Analysis—For ϵ-(γ-glutamyl)lysine and hydroxyproline analysis, OK cells were grown in 10-cm Petri dishes and solubilized with 1 ml of 0.1% (w/v) sodium deoxycholate-2 mm EDTA (DOC-EDTA). The DOC-EDTA-soluble fraction was kept for protein analysis (BCA assay) while the insoluble residue, remaining on the plate and predominantly representing the ECM, was partially digested with 0.2 mg/ml trypsin/1 mm EDTA and then scraped off. The fraction was then freeze-dried. For tTg and fibronectin analysis, cells were seeded in 96-well plates, and the DOC-EDTA-insoluble fraction was immunoprobed directly on the plate. ϵ-(γ-Glutamyl)lysine Levels—Freeze-dried proteins were suspended in 0.1 m ammonium carbonate and subjected to exhaustive proteolysis as previously described (23Griffin M. Wilson J. Mol. Cell. Biochem. 1984; 58: 37-49Crossref PubMed Scopus (42) Google Scholar). Digests were freeze-dried again and resuspended in 0.1 n HCl. ϵ-(γ-glutamyl)lysine was analyzed by cation exchange chromatography using an LKB 4151 amino acid analyzer (Amersham Biosciences) using a modification of a lithium citrate buffer method with an Ultrapac 8 cation exchange resin (8 ± 0.5-μm particle size (23Griffin M. Wilson J. Mol. Cell. Biochem. 1984; 58: 37-49Crossref PubMed Scopus (42) Google Scholar)). The detection of amino acids and peptides was undertaken by a post column reaction with 600 mg/liter o-phthalaldehyde/5 ml/liter 2-mercaptoethanol, and the fluorescence was observed at 450 nm after excitation at 360 nm using an LS1 detector (PerkinElmer Life Sciences) with analysis of chromatograms by a Nelson 9000 A-D integrator and software (Nelson Analytical). The amount of ϵ-(γ-glutamyl)lysine in each sample was quantified by standard addition of 1 nmol of ϵ-(γ-glutamyl)lysine dipeptide. Quantitation of Hydroxyproline—DOC-EDTA-insoluble proteins were acid-hydrolyzed in 6 n HCl overnight at 110 °C in an oxygen-free environment. After freeze drying and re-suspending in H2O the hydrolysates were assayed for hydroxyproline by derivatization with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and separation by reverse phase high-performance liquid chromatography (Beckman Instruments, High Wycombe, UK) (24Campa J.S. McAnulty R.J. Laurent G.J. Anal. Biochem. 1990; 186: 257-263Crossref PubMed Scopus (53) Google Scholar). Quantification was by absorption at 495 nm and peak integration as above. Extracellular Fibronectin and tTg—DOC-EDTA-insoluble proteins were blocked with milk-Tween-PBS before the addition of either 0.2 μg/ml of the monoclonal anti-tTg antibody CUB7042 or a 1:1000 dilution of rabbit anti-fibronectin (clone IST-1, Sigma) at 4 °C overnight (22Verderio E. Gaudry C. Gross S. Smith C. Downes S. Griffin M. J. Histochem. Cytochem. 1999; 47: 1417-1432Crossref PubMed Scopus (91) Google Scholar). Primary antibody was revealed with 1.5 μg/ml goat anti-mouse IgG-horseradish peroxidase or goat anti-rabbit IgG-horseradish peroxidase conjugates before the application of 70 μg/ml TMB in phosphatecitrate urea hydrogen peroxide buffer. The reaction was stopped with 2.5 m sulfuric acid, and the absorbance was read at 450 nm. Measurement of ECM Collagen by 3H Proline Labeling—Cells were seeded in 10-cm Petri dishes at a density of 3.75 × 106 per dish. Deposited collagens were labeled by the addition of 20 μl of [2,3-3H]proline (1.0 mCi/ml, ICN) at the time of plating. After 96 h the media was removed, and cells were washed with PBS. Cells were then removed with 2 ml of 0.25 m ammonium hydroxide in 50 mm Tris, pH 7.4, at 37 °C for 10 min. The soluble fraction was collected, and protein concentration was determined using the BCA assay. The dishes were washed extensively with increasing volumes of PBS before the ECM was solubilized with 2 ml of 2.5% (w/v) SDS in 50 mm Tris, pH 6.8. The dish was then scraped to ensure complete removal of the ECM, and 200 μl was measured for radioactivity in a scintillation counter. Collagen Fibrillogenesis Assay—Collagen fibrillogenesis was monitored using a spectrophotometric method (25Nomura Y. Takahashi K. Shirai K. Wada K. Agric. Biol. Chem. 1989; 53: 1614-1620Google Scholar). Type III collagen (Sigma C3511) was solubilized in 0.2 m acetic acid at a concentration of 5 mg/ml at 4 °C with constant stirring for 24 h. Initiation of fibrillogenesis was by neutralization of the collagen mixture by the addition of 10× Dulbecco's modified Eagle's medium and 0.2 m HEPES buffer to final concentrations of 1× and 0.02 m, respectively. Addition of CaCl2 to 5 mm and dithiothreitol to 5 mm was made immediately after neutralization and before addition of guinea pig liver tissue transglutaminase (Sigma T5398, ∼1000 units/mg). Fibril formation after neutralization was monitored by measuring absorbance at 325 nm using a PYE Unicam SP1800 UV spectrophotometer. mRNA Analysis—Total RNA was extracted from at least 5 × 106 cells using TRIzol™ (Invitrogen) and quantitated by optical density at 260 nm. 15 μg of total RNA was then run on a 1.2% (w/v) agarose/MOPS/formaldehyde gel and then capillary blotted on to Nylon+ (Roche Applied Science) and cross-linked with 70 mJ/cm2 UV radiation (UV cross-linker, Amersham Biosciences). This was then probed with [α-32P]dCTP random primed labeled (Prime a gene, Promega, UK), sequence-specific DNA probes for tTg (7Johnson T.S. Skill N.J. El Nahas A.M. Oldroyd S.D. Thomas G.L. Douthwaite J.A. Haylor J.L. Griffin M. J. Am. Soc. Nephrol. 1999; 10: 2146-2157Crossref PubMed Google Scholar), collagens III (26Virolainen P. Perala M. Vuorio E. Aro H.T. Clin. Orthop. 1995; 317: 263-272PubMed Google Scholar) and IV (27Kurkinen M. Condon M.R. Blumberg B. Barlow D.P. Quinones S. Saus J. Pihlajaniemi T. J. Biol. Chem. 1987; 262: 8496-8499Abstract Full Text PDF PubMed Google Scholar), fibronectin (28Schwarzbauer J.E. Tamkun J.W. Lemischka I.R. Hynes R.O. Cell. 1983; 35: 421-431Abstract Full Text PDF PubMed Scopus (483) Google Scholar), and TGFβ1 (29Millan F.A. Denhez F. Kondaiah P. Akhurst R.J. Development. 1991; 111: 131-143PubMed Google Scholar) before exposure to BioMax MS film. The resulting autoradiographs were then quantified by scanning densitometry using a Bio-Rad GS-690 densitometer and Molecular Analyst version 4 software. Transcript size was determined by comparison to RNA molecular weight markers (Promega, UK) using the same analysis package. Values were then corrected for loading using repeat probings with GAPDH. Detection of Active TGFβ—Active and total TGFβ (heat-activated) was determined using the mink lung epithelial cell assay whereby mink lung epithelial cells are stably transfected with a construct consisting of the TGFβ1 promoter region of plasmin activator inhibitor ligated to the luciferase reporter construct (30Abe M. Harpel J.G. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (676) Google Scholar). Media (serum-free) from OK cells grown under increased glucose concentrations was collected and applied to mink lung epithelial cells overnight. The media was then removed, and the cells were lysed and assayed for luciferase activity using a luciferase assay kit (Promega) and luminometer (Anthos Lab Tec Instruments, Salzberg, Austria), as per the manufacturer's instructions. Commercially available TGFβ1 was used as a positive control. Collagen Secretion—Cells were grown for 96 h with [2,3-3H]proline (Amersham Biosciences) added to the culture media at a final concentration of 75 kBq/ml. To determine the total amount of [2,3-3H]proline-labeled proteins present in the media, 25 μl of media was precipitated onto 3MM filter paper using ice-cold 10% trichloroacetic acid, washed three times in 5% trichloroacetic acid and twice in ethanol before counting in a scintillation counter. To determine the collagen component of this, a parallel sample of media was digested with 30 units/ml of chromatography-purified (i.e. free of proteases and clostripain) bacterial collagenase (Sigma) for 15 min at 37 °C in 50 mm Tris, pH 7.4, 10 mm CaCl2, 0.2 m NaCl before precipitation with trichloroacetic acid. The overall amount of collagen-labeled material in the media was calculated by subtracting the counts following collagenase digestion from the total labeled protein. Fluorography of Secreted Collagens—Media from cells grown in 75 kBq/ml labeled proline (Amersham Biosciences) for 96 h was precipitated using 4 volumes of acetone, washed 3×, and then resuspended in 1% (w/v) SDS, 50 mm Tris, pH 7.4, 1 mm EDTA. 50 μl of protein was then separated on 10% (w/v) polyacrylamide gels using an interrupted SDS-PAGE (31Sykes B. Puddle B. Francis M. Smith R. Biochem. Biophys. Res. Commun. 1976; 72: 1472-1480Crossref PubMed Scopus (456) Google Scholar, 32Sykes B. Francis M.J. Smith R. Puddle B. Francis M. N. Engl. J. Med. 1977; 296: 1200-1203Crossref PubMed Scopus (94) Google Scholar) approach to allow clear separation of collagen I and III bands. The gel was then dried under vacuum at 80 °C and exposed to BioMax MS film at –70 °C for 4 weeks. Data Analysis—All data are shown as means ± S.E. Experimental groups were compared using Student's t test with unequal variance. p < 0.05 was taken as statistically significant. Effect of High Glucose on Cell Proliferation and Viability— Increasing the concentration of glucose from 6 to 36 mm in the culture media of OK PTCs over a 96-h period caused a dose-dependent increase in viable cell numbers with no significant changes observed in cell leakage (lactate dehydrogenase ratio) and cell viability (Trypan Blue) (Table I).Table IEffect of high glucose on cell proliferation and viabilityExtracellular d-glucoseCell numberTotal proteinTotal DNAIntracellular d-glucoseLDH ratioTrypan blue stainingmm× 106mg/plateμg/plateμm%61.45 ± 0.150.23 ± 0.0618.2 ± 1.30.87 ± 0.1896.6 ± 0.495.4 ± 2.17241.59 ± 0.17ap < 0.050.34 ± 0.08ap < 0.0519.9 ± 0.71.36 ± 0.1997.3 ± 0.896.6 ± 1.3361.83 ± 0.30ap < 0.050.38 ± 0.1ap < 0.0522.9 ± 2.3ap < 0.051.79 ± 0.1997.4 ± 0.794.6 ± 2.8a p < 0.05 Open table in a new tab Effect of High Glucose on Transglutaminase—Increasing the media concentration of glucose caused a dose-dependent increase in the cellular activity of Tg in OK cells (Fig. 1a). This was shown to be due to increases in tTg protein measured by both ELISA (Fig. 1b) and Western blotting (Fig. 1c), which was mRNA-dependent (Fig. 1d). Immunoprobing of cells for tTg showed that large increases in antigen were localized to cell clusters (Fig. 1e). Measurement of Tg activity and tTg antigen in the culture media demonstrated that changes in intracellular tTg were carried through to the extracellular environment with Tg activity increasing from 1.65 ± 0.21 to 2.5 ± 0.27 units/106 cells and tTg antigen from 1.51 ± 0.07 to 3.3 ± 0.41 ng/106 cells as glucose concentration was increased from 6 to 36 mm, respectively. Effects of High Glucose on the Extracellular Matrix—Following the removal of cells using a deoxycholate-EDTA solution (DOC-EDTA), the remaining extracellular proteins (i.e. ECM) increased in response to glucose from 1.1 ± 0.1 mg/107 cells at 6 mm glucose to 2.5 ± 0.2 mg/107 cells at 36 mm glucose (Fig. 2a). Analysis of fibronectin (Fig. 2b) and total collagen (hydroxyproline) (Fig. 2c) showed that both these proteins were in part responsible for increased levels of ECM proteins. Northern blot analysis indicated that these increases were mRNA-dependent with specific changes in fibronectin, collagen I and IV mRNA levels (Fig. 2, d–f). There was no measurable change in TGFβ1 mRNA or active TGFβ1 present in the cell culture medium (data not shown) suggesting these i" @default.
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• W2147339154 date "2004-11-01" @default.
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• W2147339154 title "Inhibition of Transglutaminase Activity Reduces Extracellular Matrix Accumulation Induced by High Glucose Levels in Proximal Tubular Epithelial Cells" @default.
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• W2147339154 doi "https://doi.org/10.1074/jbc.m402698200" @default.
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