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• W1989228854 abstract "The mechanisms involved in tubular hypertrophy in diabetic nephropathy are unclear. We investigated the role of exchange protein activated by cAMP 1(Epac1), which activates Rap-family G proteins in cellular hypertrophy. Epac1 is expressed in heart, renal tubules, and in the HK-2 cell line. In diabetic mice, increased Epac1 expression was observed, and under high glucose ambience (HGA), HK-2 cells also exhibited increased Epac1 expression. We isolated a 1614-bp DNA fragment upstream of the initiation codon of Epac1 gene, inclusive of glucose response elements (GREs). HK-2 or COS7 cells transfected with the Epac1 promoter revealed a dose-dependent increase in its activity under HGA. Mutations in GRE motifs resulted in decreased promoter activity. HK-2 cells exhibited a hypertrophic response and increased protein synthesis under HGA, which was reduced by Epac1-siRNA or -mutants, whereas the use of a protein kinase A inhibitor had minimal effect. Epac1 transfection led to cellular hypertrophy and increased protein synthesis, which was accentuated by HGA. HGA increased the proportion of cells in the G0/G1 cell-cycle phase, and the expression of pAkt and the cyclin-dependent kinase inhibitors p21 and p27 was increased while the activity of cyclin-dependent kinase 4 decreased. These effects were reversed following transfection of cells with Epac1-siRNA or -mutants. These data suggest that HGA increases GRE-dependent Epac1 transcription, leading to cell cycle arrest and instigation of cellular hypertrophy. The mechanisms involved in tubular hypertrophy in diabetic nephropathy are unclear. We investigated the role of exchange protein activated by cAMP 1(Epac1), which activates Rap-family G proteins in cellular hypertrophy. Epac1 is expressed in heart, renal tubules, and in the HK-2 cell line. In diabetic mice, increased Epac1 expression was observed, and under high glucose ambience (HGA), HK-2 cells also exhibited increased Epac1 expression. We isolated a 1614-bp DNA fragment upstream of the initiation codon of Epac1 gene, inclusive of glucose response elements (GREs). HK-2 or COS7 cells transfected with the Epac1 promoter revealed a dose-dependent increase in its activity under HGA. Mutations in GRE motifs resulted in decreased promoter activity. HK-2 cells exhibited a hypertrophic response and increased protein synthesis under HGA, which was reduced by Epac1-siRNA or -mutants, whereas the use of a protein kinase A inhibitor had minimal effect. Epac1 transfection led to cellular hypertrophy and increased protein synthesis, which was accentuated by HGA. HGA increased the proportion of cells in the G0/G1 cell-cycle phase, and the expression of pAkt and the cyclin-dependent kinase inhibitors p21 and p27 was increased while the activity of cyclin-dependent kinase 4 decreased. These effects were reversed following transfection of cells with Epac1-siRNA or -mutants. These data suggest that HGA increases GRE-dependent Epac1 transcription, leading to cell cycle arrest and instigation of cellular hypertrophy. Exchange protein directly activated by cAMP (Epac1) is a novel cAMP-activated guanine nucleotide exchange factor (GEF) for Ras-like GTPases, such as Rap1,1Gloerich M. Bos J.L. Epac: defining a new mechanism for cAMP.Annu Rev Pharmacol. 2005; 50: 355-375Crossref Scopus (381) Google Scholar, 2de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.Nature. 1998; 396: 474-477Crossref PubMed Scopus (1617) Google Scholar which cycle between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. GEFs, such as Epac1, catalyze the exchange of GDP for the more abundant GTP, and thus activate Rap1-GTP binding protein.3Takai Y. Sasaki T. Matozaki T. Small GTP-binding proteins.Physiol Rev. 2001; 81: 153-208Crossref PubMed Scopus (2057) Google Scholar The Rap1 regulates diverse pivotal cellular processes, including cell survival, proliferation, differentiation, hypertrophy, intracellular vesicular trafficking, cytoskeletal rearrangement, cell cycle events, and glucose transport.1Gloerich M. Bos J.L. Epac: defining a new mechanism for cAMP.Annu Rev Pharmacol. 2005; 50: 355-375Crossref Scopus (381) Google Scholar, 2de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.Nature. 1998; 396: 474-477Crossref PubMed Scopus (1617) Google Scholar, 3Takai Y. Sasaki T. Matozaki T. Small GTP-binding proteins.Physiol Rev. 2001; 81: 153-208Crossref PubMed Scopus (2057) Google Scholar, 4Evans T. Hart M.J. Cerione R.A. Ras superfamilies: regulatory proteins and post-translational modifications.Curr Opin Cell Biol. 1991; 2: 185-191Crossref Scopus (17) Google Scholar Although Epac1 participates in gene transcription, insulin secretion, and ion transport,1Gloerich M. Bos J.L. Epac: defining a new mechanism for cAMP.Annu Rev Pharmacol. 2005; 50: 355-375Crossref Scopus (381) Google Scholar more recent studies have suggested Epac proteins may regulate the development of cardiac hypertrophy.5Roscioni S.S. Elzinga C.R. Schmidt M. Epac: effectors and biological functions.Naunyn Schmiedebergs Arch Pharmacol. 2008; 377: 345-357Crossref PubMed Scopus (122) Google Scholar Although, a related cAMP–protein kinase A (PKA) pathway modulates a number of different physiological and pathological processes, including regulation of a cell cycle, ion transport, cellular proliferation, and extracellular matrix expression in normal kidney and in various chronic kidney diseases,6Cheng X. Ji Z. Tsalkova T. Mei F. Epac and PKA: a tale of two intracellular cAMP receptors.Acta Biochim Biophys Sin (Shanghai). 2008; 40: 651-662Crossref PubMed Scopus (258) Google Scholar, 7Marfella-Scivittaro C. Quinones A. Orellana S.A. cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia.Am J Physiol Cell Physiol. 2002; 282: C693-C707Crossref PubMed Scopus (20) Google Scholar the role of Epac1 in renal pathophysiology has been delineated to a limited extent, regulating intracellular Ca2+ mobilization and apical exocytotic insertion of AQP2 in inner medullary collecting ducts (IMCD).8Yip K.P. Epac-mediated Ca(2+) mobilization and exocytosis in inner medullary collecting duct.Am J Physiol Renal Physiol. 2006; 291: F882-F890Crossref PubMed Scopus (75) Google Scholar However, there is no available literature report describing the role of Epac1 in the progression of diabetic nephropathy. Diabetic nephropathy is now recognized as the most common cause of end-stage renal disease and accounts for 30% to 40% of all patients requiring renal replacement therapy, and hyperglycemia is implicated as a major factor in its pathogenesis.9Kanwar Y.S. Sun L. Xie P. Liu F.-Y. Chen S. A glimpse of various pathogenetic mechanisms of diabetic nephropathy.Annu Rev Pathol: mechanisms of Disease. 2011; 6: 395-423Crossref PubMed Scopus (502) Google Scholar A number of pathophysiologic mechanisms linking hyperglycemia to the development of nephropathy have been proposed and defined regarding glomerular pathobiology.10Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 200, 414:813-820Google Scholar, 11Susztak K. Bottinger E.P. Diabetic nephropathy: a frontier for personalized medicine.J Am Soc Nephrol. 2006; 17: 361-367Crossref PubMed Scopus (74) Google Scholar, 12Wolf G. Ziyadeh F.N. Cellular and molecular mechanisms of proteinuria in diabetic nephropathy.Nephron Physiol. 2007; 106: 26-31Crossref PubMed Scopus (199) Google Scholar, 13Villeneuve L.M. Natarajan R. The role of epigenetics in the pathology of diabetic complications.Am J Physiol Renal Physiol. 2010; 299: F15-F25Crossref Scopus (255) Google Scholar, 14Sharma K. Jin Y. Guo J. Ziyadeh F.N. Neutralization of TGF-β by anti-TGF-β antibody attenuates kidney hypertrophy and enhanced ECM expression in streptozotocin-induced diabetic mice.Diabetes. 1996; 45: 522-530Crossref PubMed Scopus (0) Google Scholar, 15Kashihara N. Haruna Y. Kondetti V.K. Kanwar Y.S. Oxidative stress in diabetic nephropathy.Curr Med Chem. 2010; 17: 4256-4269Crossref PubMed Scopus (353) Google Scholar The well-known characteristic structural features of renal pathology include glomerular hypertrophy, mesangial cell proliferation, podocytes loss, glomerular basement membrane thickening, and amassing of extracellular matrix in the mesangium.9Kanwar Y.S. Sun L. Xie P. Liu F.-Y. Chen S. A glimpse of various pathogenetic mechanisms of diabetic nephropathy.Annu Rev Pathol: mechanisms of Disease. 2011; 6: 395-423Crossref PubMed Scopus (502) Google Scholar, 16Mason R.M. Wahab N.A. Extracellular matrix metabolism in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: 1358-1373Crossref PubMed Scopus (521) Google Scholar Recent studies over the last decade have also linked hyperglycemia to the pathobiology of the tubulointerstitium, and injury to the latter has been known to also correlate with the degree of compromise in renal functions.17Nath K.A. Tubulointerstitial changes as a major determinant in the progression of renal damage.Am J Kidney. 1992; 20: 1-7PubMed Scopus (860) Google Scholar, 18Nath K.A. The tubulointerstitium in progressive renal disease.Kidney Int. 1998; 54: 992-994Crossref PubMed Scopus (159) Google Scholar The tubulointerstitial pathology includes tubular hypertrophy, thickening and reduplication of the tubular basement membrane and ensuing tubulointerstitial fibrosis, leading ultimately to progressive decline in renal dysfunctions.9Kanwar Y.S. Sun L. Xie P. Liu F.-Y. Chen S. A glimpse of various pathogenetic mechanisms of diabetic nephropathy.Annu Rev Pathol: mechanisms of Disease. 2011; 6: 395-423Crossref PubMed Scopus (502) Google Scholar, 16Mason R.M. Wahab N.A. Extracellular matrix metabolism in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: 1358-1373Crossref PubMed Scopus (521) Google Scholar A large array of genes that are directly related to the glomerular pathobiology has been implicated in the pathogenesis of diabetic nephropathy.10Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 200, 414:813-820Google Scholar, 11Susztak K. Bottinger E.P. Diabetic nephropathy: a frontier for personalized medicine.J Am Soc Nephrol. 2006; 17: 361-367Crossref PubMed Scopus (74) Google Scholar, 12Wolf G. Ziyadeh F.N. Cellular and molecular mechanisms of proteinuria in diabetic nephropathy.Nephron Physiol. 2007; 106: 26-31Crossref PubMed Scopus (199) Google Scholar, 13Villeneuve L.M. Natarajan R. The role of epigenetics in the pathology of diabetic complications.Am J Physiol Renal Physiol. 2010; 299: F15-F25Crossref Scopus (255) Google Scholar, 14Sharma K. Jin Y. Guo J. Ziyadeh F.N. Neutralization of TGF-β by anti-TGF-β antibody attenuates kidney hypertrophy and enhanced ECM expression in streptozotocin-induced diabetic mice.Diabetes. 1996; 45: 522-530Crossref PubMed Scopus (0) Google Scholar, 15Kashihara N. Haruna Y. Kondetti V.K. Kanwar Y.S. Oxidative stress in diabetic nephropathy.Curr Med Chem. 2010; 17: 4256-4269Crossref PubMed Scopus (353) Google Scholar Some of these may be relevant to the pathobiology of tubulointerstitium as well. By subtractive hybridization, a handful of genes have been identified that may be relevant to the pathobiology of tubulointerstitium in diabetic nephropathy,19Wada J. Kanwar Y.S. Characterization of mammalian translocase of inner mitochondrial membrane (Timm44) isolated from diabetic newborn mouse kidney.Proc Natl Acad Sci USA. 1998; 95: 144-149Crossref PubMed Scopus (61) Google Scholar, 20Lin S. Chugh S. Pan X. Wallner E.I. Wada J. Kanwar Y.S. Identification of up-regulated Ras-like GTPase Rap1b, by suppressive subtractive hybridization.Kidney Int. 2001; 60: 2129-2141Crossref PubMed Scopus (12) Google Scholar among them the target of Epac1, Rap1b G-protein,.21Lin S. Sahai A. Chugh S.S. Pan X. Wallner E.I. Danesh F.R. Lomasney J.W. Kanwar Y.S. High glucose stimulates synthesis of fibronectin via a novel protein kinase C Rap1b, and B-Raf signaling pathway.J Biol Chem. 2002; 277: 41725-41735Crossref PubMed Scopus (46) Google Scholar But which of these genes are relevant to the tubular hypertrophy in early stages of diabetic nephropathy? Having delineated the role Rap1b in the pathogenesis of diabetic nephropathy21Lin S. Sahai A. Chugh S.S. Pan X. Wallner E.I. Danesh F.R. Lomasney J.W. Kanwar Y.S. High glucose stimulates synthesis of fibronectin via a novel protein kinase C Rap1b, and B-Raf signaling pathway.J Biol Chem. 2002; 277: 41725-41735Crossref PubMed Scopus (46) Google Scholar and the literature information suggesting the role Epac1 in cardiac myocyte hypertrophy,22Morel E. Marcantoni A. Gastineau M. Birkedal R. Rochais F. Garnier A. Lompre A.M. Vandecasteele G. Lezoualc'h F. cAMP-binding protein Epac induces cardiomyocyte hypertrophy.Circ Res. 2005; 97: 1296-1304Crossref PubMed Scopus (156) Google Scholar, 23Ulucan C. Wang X. Baljinnyam E. Bai Y. Okumura S. Sato M. Minamisawa S. Hirotani S. Ishikawa Y. Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy.Am J Physiol Heart Circ Physiol. 2007; 293: H1662-H1672Crossref PubMed Scopus (78) Google Scholar modulated via β-adrenergic receptors in a protein kinase A (PKA)–independent fashion,24Metrich M. Lucas A. Gastineau M. Samuel J.L. Heymes C. Morel E. Lezoualc'h F. Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy.Circ Res. 2008; 102: 959-965Crossref PubMed Scopus (170) Google Scholar studies were initiated to explore the relevance of Epac1 in cellular hypertrophy of tubules in diabetic nephropathy, using in vivo and in vitro approaches. A diabetic state was induced in 10-week-old CD1 mice (Harlan Co., Indianapolis, IN) by an injection of streptozotocin, STZ (200 mg/kg body wt; Sigma Chemical, St. Louis, MO). After 1 week, the mice with blood glucose levels >250 mg/dl were selected for various studies. The mice were sacrificed 8 weeks after the induction of diabetes. All of the animal procedures used in this study were approved by the Animal Care and Use Committee of Northwestern University. Various cell lines used in this investigation included the followings: HepG2 (human hepatocellular carcinoma), mouse glomerular podocyte, mouse and rat mesangial cells, HK-2 (human proximal tubular cell), rat proximal tubular cell, mIMCD-3 (mouse inner medullary collecting duct cell), LLCPK1 (porcine renal tubular cell), and COS7 (African monkey kidney cell). They were purchased from American Type Culture Collections (Manassas, VA). The cell lines were maintained in media and culture conditions recommended by the vendor, and used for various studies. Immunohistochemical and in situ hybridization studies were performed to assess the spatiotemporal expression of Epac1 in kidneys of normal and diabetic mice. In situ hybridization was performed as previously described.25Kanwar Y.S. Yang Q. Tian Y. Lin S. Wada J. Chugh S. Srivastava S.K. Relevance of renal-specific oxidoreductase in tubulogenesis during mammalian nephron development.Am J Physiol Renal Physiol. 2002; 282: F752-F762Crossref PubMed Scopus (9) Google Scholar Briefly, 1- to 2-mm-thick kidney tissue slices were dehydrated in graded series of ethanol and embedded in paraffin. Then 4-μm thick sections were prepared and mounted on HCl-treated and Vectabond-coated slides (Vector Labs, Burlingame, CA). The sections were hybridized with Epac1 digoxigenin (DIG)-labeled RNA sense and antisense probes prepared by using in vitro transcription system (Roche Diagnostics, Indianapolis, IN). For preparation of probes, a 407 bp DNA fragment of Epac1 gene was generated by PCR using 5′-CGAGCAGGAGCACAGCACCTACATCTG-3′ (sense) and 5′-TCACTTCTCTCACCGAGGCCGTCACCG-3′ (antisense) primers. The DIG-probes were then subjected to limited alkaline hydrolysis to obtain polynucleotide fragments with a size range of 100 to 150 bp. After hybridization, the slides were successively washed with 2×, 1×, and 0.5× standard saline citrate in the presence of 1 mmol/L dithiothreitol. The tissue sections were blocked with 3% bovine serum albumin and incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. They were then developed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate solution (Roche Diagnostics). For immunohistochemical studies avidin-biotin complex method (Vector Labs) was used. The 4-μm thick kidney sections were deparaffinized and hydrated in graded series of decreasing concentrations of ethanol. They were then successively incubated with rabbit anti-Epac1 (Abcam, Cambridge, MA), biotinylated anti-rabbit IgG, and streptavadin conjugated with horseradish peroxidase (HRP) in between brief washes of PBS. The sections were then treated with SIGMAFAST 3′,3′-diamino-benzidine solution for 3 to 5 minutes at 22°C to develop the HRP reaction product. The sections were counterstained with hematoxylin, coverslip mounted, and examined. Northern and Western blot analyses were performed to assess the Epac1 expression in control and diabetic animals. For Northern analysis, total RNA was prepared from mice kidneys and other tissues by the acid guanidinium isothiocyanate-phenol-chloroform extraction method.26Chomcznski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium isothiocynate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159PubMed Google Scholar About 20 μg of total RNAs extracted from kidneys of diabetic and control mice was glyoxylated, subjected to 1% agarose gel electrophoresis, and then capillary-transferred to Hybond N+ nylon membrane (GE Health care Bioscience Corp., Piscataway, NJ). After cross-linking of RNA to the membrane, prehybridization and hybridization of various membrane blots were performed with various α-[32P]-dCTP labeled (1 × 106 cpm/mL) cDNA probes of Epac1 cDNA fragment described above.25Kanwar Y.S. Yang Q. Tian Y. Lin S. Wada J. Chugh S. Srivastava S.K. Relevance of renal-specific oxidoreductase in tubulogenesis during mammalian nephron development.Am J Physiol Renal Physiol. 2002; 282: F752-F762Crossref PubMed Scopus (9) Google Scholar Following the preparation of autoradiograms, the membrane blots were stripped by boiling in 0.1% SDS buffer for 2 minutes and re-probed with radiolabeled β-actin cDNA probe. For Western blot analysis, tissue lysates were prepared by homogenizing control and diabetic kidneys in ice-cold extraction buffer (10 mmol/L HEPES/1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 1 mmol/L PMSF, pH 7.4). The homogenate was centrifuged at 10,000 × g for 30 minutes at 4°C, and the supernatant was saved. The protein concentration in the supernatant was adjusted to 2 mg/mL. Equal amounts (∼20 μg) of protein (control versus diabetic) were loaded onto the gel-wells and subjected to 10% SDS/PAGE under reducing conditions. The gel proteins were electroblotted onto a nitrocellulose membrane. The membrane was immersed in a blocking solution containing 5% nonfat milk in Tris-buffered saline-T (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween-20) followed by successive 60-minute incubations with Epac1antibody and goat anti-rabbit IgG conjugated with HRP at 37°C. The membrane was washed three times with Tris-buffered saline-T and autoradiogram developed using the SuperSignal West Pico Chemilumniscent kit (Thermo Scientific, Rockford, IL). First, RT-PCR analyses were performed to assess the Epac1 expression in various liver and kidney cell lines. The primers used for various mammalian species were as follows. Epac1-Human: 5′-TTCATGAGGGAAACCACACA-3′ (sense), 5-CCTTCAGCTGCTGGACATAA-3′ (antisense), (product size, 246 bp); Epac1-Mouse: 5′-CTGCTGCTCAAAGACGTGAC-3′, (sense) 5′-GACTGCTCAGAACACGTG GA-3′ (antisense) (product size, 218 bp); Epac1-Rat: 5′-TTCATGAAGGGAACCACACA-3′ (sense), 5′-GCTCGGAACATGTGGAGA TT-3′ (antisense) (product size, 189 bp). The proximal tubular cell line, HK-2, was used for further studies because the morphological studies indicated a relatively high expression in the renal proximal tubules. The HK-2 cells were maintained in a defined medium [3:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), HEPES (14 mmol/L)] at 37°C. After achieving 80% confluency, the medium was changed to FBS-free DMEM. Varying concentrations of D-glucose (5 to 30 mmol/L) was added and cell culture maintained for 48 to 72 hours. L-glucose served as a control. The cells were then processed for Northern and Western blot analyses as described above. To identify the minimal promoter region and to understand the mechanism(s) of glucose-induced up-regulation of Epac1, the GreatEscApe SEAP system (Clonetech, Mountain View, CA) was used. First, a ∼1.6 kb DNA fragment upstream of 5′ flanking region of Epac1 cDNA was generated by PCR and cloned into pCRII vector (Invitrogen, Carlsbad, CA). This DNA fragment was used as a template to generate four deletion constructs (−1 to −1547, −1 to −1163, −1 to −931, and −1 to −529). For generation of the constructs, four sense primers and one antisense primer were synthesized (Integrated DNA Technologies, Skokie, IL). Their sequences were as follows: Epac-G1-SE (5′-CGAAGCTTGAAACTCTGCAACAAATCCG-3′), Epac-G2-SE (5′-CGCTGAAGCTTTCCCTAGTTTCCTTGTTTG-3′), Epac-G3-SE (5′-CTCTGAAGCTTTAAAGCTCGTGCCTGCTC-3′), Epac-G4-SE (5′-CGCTGAAGC TTCGGAGTCAGGGCCAAAGAA-3′) and Epac-G1-AS (5′-GCAGCCTCGAGGAACACTAGCTGGTAAGAACAGCA-3′). HindIII (AAG CTT) and XhoI (CTC GAG) sites (italicized) were included in the sense and antisense primers, respectively. Using these primers and pCRII/EpacI DNA plasmid as a template, various PCR products were generated and subcloned into XhoI- and HindIII-digested pSEAP2-Enhancer plasmid vector (Clonetech, Mountain View, CA) and sequenced. The minimal promoter activity of Epac1 gene was measured 48 hours following transfection of various cell lines (HK-2, LLCPK1 and COS7) with plasmids containing different fragments of the promoter using a GreatEscAPe SEAP fluorescence detection kit (Clonetech, Mountain View, CA), as previously described.27Sun L. Pan X. Wada J. Haas C.S. Wuthrich R.P. Danesh F.R. Chugh S.S. Kanwar Y.S. Isolation and functional analyses of mouse UbA52 gene and its relevance to diabetic nephropathy.J Biol Chem. 2002; 277: 29953-29962Crossref PubMed Scopus (24) Google Scholar For transfection, Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent kit was used. The activities of various deletion constructs were expressed as the percentages of the activity in the deletion construct with the highest promoter activity, which was designated as being100%. The promoter region of Epac1 contains two glucose responsive element (GRE) (CACGTG) sites located at −1112 to −1106 (GRE1) and −479 to −473 (GRE2), flanking the open reading frame of Epac1 gene. To assess whether the GRE are functional, COS7 cells were exposed to different concentrations of D-glucose (5 to 35 mmol/L) and then transfected with Epac1 gene promoter plasmid containing largest DNA fragment (ie, deletion construct # 1 [DC1]).27Sun L. Pan X. Wada J. Haas C.S. Wuthrich R.P. Danesh F.R. Chugh S.S. Kanwar Y.S. Isolation and functional analyses of mouse UbA52 gene and its relevance to diabetic nephropathy.J Biol Chem. 2002; 277: 29953-29962Crossref PubMed Scopus (24) Google Scholar To confirm GRE's role in the transcription of Epac1 expression under high glucose ambience, GRE1 and GRE2 were modified (GRE1: CACGTG to CAGCTG; GRE2: CACGTG to CAAGTG), using site-directed mutagenesis kit following vendor's instructions (Stratagene, La Jolla, CA). They were then used for the SEAP activity analysis, as described above following their subcloning into the pSEAP2-Enhancer plasmid vector. Two human Epac1 mammalian expression vector plasmid constructs were a kind gift of Dr. Johannes Bos (University Medical Centre, Utrecht, The Netherlands). The first one included full-length Epac1 cDNA, and it was designated as pMT2-HA-Epac1. The second one lacked the first 322 amino acids, such that the cAMP binding site is deleted, and it was designated as pMT2-HA-Epac1ΔcAMP or Epac1 mutant1 (Epac1-M1). Another Epac1 construct was generated by inserting a stop codon after the glutamate residue at position 614 using site-directed mutagenesis kit (Stratagene), such that the guanine exchange factor (GEF) domain is deleted, and it was designated as pMT2-HA-Epac1ΔGEF or Epac1 mutant2 (Epac1-M2). A cell-permeable cAMP analog, 8-CPT-2-O-Me-cAMP (8-cAMP) that selectively binds to Epac1 and triggers Epac1 signaling was purchased from Sigma Chemicals. Epac1 siRNA, that has been shown to knock down the Epac1 signaling28Birukova A.A. Burdette D. Moldobaeva N. Xing J. Fu P. Birkov K.G. Rac GTPase is a hub for protein kinase A and Epac signaling in endothelial barrier protection by cAMP.Microvasc Res. 2010; 79: 128-138Crossref PubMed Scopus (65) Google Scholar and siRNA transfection transmessenger kit were obtained from Qiagen company (Invitrogen, Carlsbad, CA). The HK-2 cells were maintained in a defined medium at 37°C. After achieving 80% confluency, the culture was replaced with fresh defined medium containing 1% fetal bovine serum. The cells were exposed to 5 to 30 mmol/L D-glucose for 48 hours. In various experiments the cells were either pretreated with cAMP analog or H89, PKA inhibitor, or transfected with Epac1 or its mutants, Epac1-M1 and Epac1-M2, or Epac1 siRNA. The cells were then processed for [3H] leucine incorporation, measurement of protein and DNA contents and their ratio, and also for confocal microscopy to assess the morphology. Three controls were included for these experiments, which included cells subjected to low glucose (5 mmol/L) ambience, and cells transfected either with scrambled oligo or empty pMT2-HA vector. For protein synthesis, reflective of cellular hypertrophy, the treated cells were pulsed with [3H] leucine (10 μCi/mL) for 12 hours. The cells were harvested and washed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS). After counting the cells with a hemocytometer, a cell pellet was prepared by sedimentation in an Eppendorf microfuge (Eppendorf AG, Hamburg, Germany) for 1 minute. The pellet was dissolved in 1 N NaOH for 30 minutes with intermittent vortexing, followed by another centrifugation for 1 minute. Then 10% TCA was added to the supernatant and it was kept at 4°C for 10 minutes. Finally, TCA-precipitated, incorporated radioactivity was determined by liquid scintillation counting and expressed as 1 × 106 cpm/103 cells. For protein and DNA measurements, HK-2 cells were maintained in 12-well culture plates. At the termination of the experiment, the cells from each well were lysed with 100 μl of PBS containing 0.003% digitonin and 10 ng/mL of ribonuclease A for 10 minutes at 22°C, followed by addition of 50 μl of 0.8 M urea. For protein assay a 20 μl aliquot of the lysate was mixed with 200 μl of nano-orange reagent from NanoOrange protein quantitation kit (Invitrogen). The samples were then transferred to microplates and readings recorded with a fluorometer set at 485 nm (excitation) and 590 nm (capture) wavelengths. The readings were then read against bovine serum albumin standards, following vendor's instructions. Similarly, another 20 μl aliquot was used for measuring DNA concentration by using a Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen). The absorbance in the samples was recorded at 260 nm. The values were read against dsDNA standards and DNA concentration determined. Finally, protein/DNA ratio was calculated for six samples for each experimental variable. To measure the cell surface area, HK-2 cells were stained with rhodamine phalloidin (1:50 dilution) for 20 minutes to visualize F-actin. They were then counterstained with 300 nmol/L DAPI at 22°C for 1 minute and briefly washed with PBS. After placing a drop of Antifade solution (Invitrogen) the cells were coverslip mounted. Confocal microscopy was performed with an LSM 510 META laser scanning microscope (Zeiss, Thornwood, NY). The following wavelengths were used for excitation: 488 nm (green), 543 nm (red), and 405 nm (blue). LSM 510 software was used to measure the cell surface area. The data were derived from at least ten randomly selected fields from six separate experiments. The HK-2 cells were maintained in 100 mm culture dishes as described above. They were then exposed to 5 to 30 mmol/L D-glucose for 72 hours. Various experimental manipulations included the pretreatment with cAMP analog, or transfection with Epac1 or its mutant, Epac1-M1, or Epac1 siRNA. The cells were processed for fluorescence-activated cell sorter (FACS) and Western blot analyses, and measurement of CDK4 kinase activity. For FACS analyses, the cells were treated with 0.05% trypsin in PBS buffer containing 0.05% EDTA. Following which, single cell suspension (2 × 106 cells/mL) in PBS buffer was prepared and fixed with 70% ethanol for 1 hour at 4°C. The fixed cells were washed with PBS and stained with propidium iodide (PI, 50 μg/mL) buffer containing, 500 μg/mL DNase-free RNase (500 μg/mL) and 0.1% Triton X-100 for 3 hours at 4°C. The samples were then analyzed by BD FACS Canto II flow Cytometer (BD Biosciences, San Jose, CA). The expression of various cell cycle proteins was evaluated by Western blot analyses. The immunoblots were prepared from extracts of HK-2 cells subjected to various treatments as described above. They were probed with the following antibodies: anti-Epac1, anti –AKT, anti-phospho-AKT, anti -p21, anti -p27, and anti-β-actin (Cell Signaling Technologies, Danvers, MA). For measuring the kinase activity of CDK4, cellular extracts from HK-2 cells that had undergone various treatments" @default.
• W1989228854 created "2016-06-24" @default.
• W1989228854 creator A5005367596 @default.
• W1989228854 creator A5024912124 @default.
• W1989228854 creator A5030494055 @default.
• W1989228854 creator A5067471791 @default.
• W1989228854 creator A5073206138 @default.
• W1989228854 date "2011-10-01" @default.
• W1989228854 modified "2023-10-02" @default.
• W1989228854 title "Epac1-Mediated, High Glucose–Induced Renal Proximal Tubular Cells Hypertrophy via the Akt/p21 Pathway" @default.
• W1989228854 cites W1509669696 @default.
• W1989228854 cites W1885709862 @default.
• W1989228854 cites W1963864987 @default.
• W1989228854 cites W1968648927 @default.
• W1989228854 cites W1972365461 @default.
• W1989228854 cites W1981853910 @default.
• W1989228854 cites W1987979328 @default.
• W1989228854 cites W1995978677 @default.
• W1989228854 cites W2000998194 @default.
• W1989228854 cites W2015883661 @default.
• W1989228854 cites W2032174196 @default.
• W1989228854 cites W2033863425 @default.
• W1989228854 cites W2039870127 @default.
• W1989228854 cites W2044437167 @default.
• W1989228854 cites W2050332600 @default.
• W1989228854 cites W2050843664 @default.
• W1989228854 cites W2050864381 @default.
• W1989228854 cites W2051867467 @default.
• W1989228854 cites W2052520208 @default.
• W1989228854 cites W2054017727 @default.
• W1989228854 cites W2055943329 @default.
• W1989228854 cites W2059600600 @default.
• W1989228854 cites W2060863299 @default.
• W1989228854 cites W2061202476 @default.
• W1989228854 cites W2061588741 @default.
• W1989228854 cites W2063443944 @default.
• W1989228854 cites W2064577372 @default.
• W1989228854 cites W2070602138 @default.
• W1989228854 cites W2080505183 @default.
• W1989228854 cites W2086999880 @default.
• W1989228854 cites W2088512240 @default.
• W1989228854 cites W2091389364 @default.
• W1989228854 cites W2092830124 @default.
• W1989228854 cites W2097158209 @default.
• W1989228854 cites W2101776890 @default.
• W1989228854 cites W2107709584 @default.
• W1989228854 cites W2111694965 @default.
• W1989228854 cites W2111778006 @default.
• W1989228854 cites W2112856354 @default.
• W1989228854 cites W2113839433 @default.
• W1989228854 cites W2119348300 @default.
• W1989228854 cites W2119903978 @default.
• W1989228854 cites W2122981222 @default.
• W1989228854 cites W2124502714 @default.
• W1989228854 cites W2126312769 @default.
• W1989228854 cites W2131083409 @default.
• W1989228854 cites W2133070723 @default.
• W1989228854 cites W2137137048 @default.
• W1989228854 cites W2149303590 @default.
• W1989228854 cites W2151223245 @default.
• W1989228854 cites W2155574603 @default.
• W1989228854 cites W2158726350 @default.
• W1989228854 cites W2159864910 @default.
• W1989228854 cites W2161009969 @default.
• W1989228854 cites W2165803538 @default.
• W1989228854 cites W2167290692 @default.
• W1989228854 cites W2313400759 @default.
• W1989228854 cites W2330039507 @default.
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