Increased Hexokinase Activity, of Either Ectopic or Endogenous Origin, Protects Renal Epithelial Cells against Acute Oxidant-induced Cell Death
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m110927200
ISSN1083-351X
AutoresJane M. Bryson, Platina E. Coy, Kathrin Gottlob, Nissim Hay, R. Brooks Robey,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoGlucose (Glc) metabolism protects cells against oxidant injury. By virtue of their central position in both Glc uptake and utilization, hexokinases (HKs) are ideally suited to contribute to these effects. Compatible with this hypothesis, endogenous HK activity correlates inversely with injury susceptibility in individual renal cell types. We recently reported that ectopic HK expression mimics the anti-apoptotic effects of growth factors in cultured fibroblasts, but anti-apoptotic roles for HKs have not been examined in other cell types or in a cellular injury model. We therefore evaluated HK overexpression for the ability to mitigate acute oxidant-induced cell death in an established epithelial cell culture injury model. In parallel, we examined salutary heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) treatment for the ability to 1) increase endogenous HK activity and 2) mimic the protective effects of ectopic HK expression. Both HK overexpression and HB-EGF increased Glc-phosphorylating capacity and metabolism, and these changes were associated with markedly reduced susceptibility to acute oxidant-induced apoptosis. The uniform Glc dependence of these effects suggests an important adaptive role for Glc metabolism, and for HK activity in particular, in the promotion of epithelial cell survival. These findings also support the contention that HKs contribute to the protective effects of growth factors. Glucose (Glc) metabolism protects cells against oxidant injury. By virtue of their central position in both Glc uptake and utilization, hexokinases (HKs) are ideally suited to contribute to these effects. Compatible with this hypothesis, endogenous HK activity correlates inversely with injury susceptibility in individual renal cell types. We recently reported that ectopic HK expression mimics the anti-apoptotic effects of growth factors in cultured fibroblasts, but anti-apoptotic roles for HKs have not been examined in other cell types or in a cellular injury model. We therefore evaluated HK overexpression for the ability to mitigate acute oxidant-induced cell death in an established epithelial cell culture injury model. In parallel, we examined salutary heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) treatment for the ability to 1) increase endogenous HK activity and 2) mimic the protective effects of ectopic HK expression. Both HK overexpression and HB-EGF increased Glc-phosphorylating capacity and metabolism, and these changes were associated with markedly reduced susceptibility to acute oxidant-induced apoptosis. The uniform Glc dependence of these effects suggests an important adaptive role for Glc metabolism, and for HK activity in particular, in the promotion of epithelial cell survival. These findings also support the contention that HKs contribute to the protective effects of growth factors. Glucose (Glc) 1The abbreviations used are: GlcglucoseDAPI4′,6′-diamidino-2-phenylindoleEGFepidermal growth factorHB-EGFheparin-binding EGF-like growth factorHBSSHanks' balanced salt solutionHKhexokinaseHsPTCHomo sapiens proximal tubule cellsLDHlactate dehydrogenasem.o.i.multiplicity of infectionMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumPIpropidium iodideGLUT1glucose transporter 1rAdrecombinant adenovirusHKIhexokinase Iwtwild-typeGSHreduced glutathione 1The abbreviations used are: GlcglucoseDAPI4′,6′-diamidino-2-phenylindoleEGFepidermal growth factorHB-EGFheparin-binding EGF-like growth factorHBSSHanks' balanced salt solutionHKhexokinaseHsPTCHomo sapiens proximal tubule cellsLDHlactate dehydrogenasem.o.i.multiplicity of infectionMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumPIpropidium iodideGLUT1glucose transporter 1rAdrecombinant adenovirusHKIhexokinase Iwtwild-typeGSHreduced glutathionemetabolism plays a critical role in the protection of a variety of cell types against oxidant-induced cell death (1.Neely J.R. 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Apoptosis. 2000; 5: 99-105Crossref PubMed Scopus (135) Google Scholar). The mechanisms underlying these cytoprotective effects are poorly understood, but contributions by both altered energy metabolism and cellular redox status have been suggested. Hexokinases (HKs) play a central role in both the uptake and utilization of Glc by catalyzing the first committed step of its metabolism, the phosphorylation of Glc to yield Glc 6-phosphate. By this mechanism, HKs maintain the downhill concentration gradient that permits facilitated Glc entry into cells. They also initiate all major pathways of Glc utilization and are ideally positioned to influence metabolic flux through both the glycolytic and the pentose phosphate pathways. Glycolytic flux is intimately linked to cellular energy metabolism, whereas pentose phosphate pathway flux plays a pivotal role in maintaining the cellular redox state. In principle, therefore, HK activity may directly contribute to the salutary effects associated with Glc metabolism in the setting of oxidative stress.Three high affinity HK isoforms, HKI, HKII, and HKIII, are expressed in the mammalian kidney (10.Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Abstract Full Text PDF PubMed Google Scholar). Of these, HKI constitutes the principal renal isoform and accounts for roughly two-thirds of the total Glc-phosphorylating capacity of the normal kidney. The remaining HK activity is equally attributable to the two lower abundance renal isoforms, HKII and HKIII. Very little is known, however, about the normal expression, regulation, or function of individual HK isoforms in specific renal cell types. Cortical HK activity is increased in a variety of conditions associated with structural or functional alterations in the kidney (11.Dubach U.C. Recant L. J. Clin. Invest. 1960; 39: 1364-1371Crossref PubMed Scopus (18) Google Scholar, 12.Anderson J.W. Stowring L. Am. J. Physiol. 1973; 224: 930-936Crossref PubMed Scopus (54) Google Scholar, 13.Steer K.A. Sochor M. Gonzalez A.-M. McLean P. FEBS Lett. 1982; 150: 494-498Crossref PubMed Scopus (25) Google Scholar, 14.Sochor M. Kunjara S. McLean P. Horm. Metabol. Res. 1988; 20: 676-681Crossref PubMed Scopus (11) Google Scholar), but the specific cell types and HK isoforms associated with these changes have not been defined. Given the substantial proximal tubular composition of the renal cortex, it is reasonable to assume that this nephron segment contributes significantly to these changes.The proximal tubule plays a number of centrally important roles in normal renal physiology. This nephron segment also exhibits marked susceptibility to both oxidative and toxic injury in vivo and represents the principal renal cell type affected by a wide variety of pathophysiologic conditions. Because a general inverse correlation exists between endogenous HK activity and injury susceptibility in individual renal cell types (15.Schmidt U. Marosvari I. Dubach U.C. FEBS Lett. 1975; 53: 26-28Crossref PubMed Scopus (40) Google Scholar, 16.Vandewalle A. Wirthensohn G. Heidrich H.-G. Guder W.G. Am. J. Physiol. 1981; 240: F492-F500PubMed Google Scholar, 17.Lawrence G.M. Trayer I.P. Histochem. J. 1984; 16: 697-708Crossref PubMed Scopus (16) Google Scholar, 18.Preller A. Wilson J.E. Arch. Biochem. Biophys. 1992; 294: 482-492Crossref PubMed Scopus (49) Google Scholar), we hypothesized that HK activity may constitute an intrinsic cellular protective mechanism and play important adaptive roles in cellular responses to oxidative stress. To directly test this hypothesis and to better understand this important family of enzymes in the kidney, we first examined the functional consequences of ectopic HK expression in an established cell culture model of acute oxidant-induced proximal tubule cell injury (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar, 20.Zager R.A. Burkhart K. Kidney Int. 1997; 51: 728-738Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 21.Zager R.A. Burkhart K.M. Kidney Int. 1998; 53: 1661-1672Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We then investigated whether changes in endogenous HK activity could contribute to the well-described salutary effects of growth factors on this cell type (22.Hammerman M.R. Safirstein R. Harris R.C. Toback F.G. Humes H.D. Am. J. Physiol. 2000; 279: F3-F11Google Scholar). Heparin-binding EGF-like growth factor (HB-EGF) was specifically selected for study on the basis of its increased renal expression during recovery from acute ischemic injury (23.Homma T. Sakai M. Cheng H.F. Yasuda T. Coffey R.J.J. Harris R.C. J. Clin. Invest. 1995; 96: 1018-1025Crossref PubMed Scopus (130) Google Scholar), as well as the demonstrated ability of the closely related family member EGF to ameliorate toxic and ischemic renal injury in vivo (22.Hammerman M.R. Safirstein R. Harris R.C. Toback F.G. Humes H.D. Am. J. Physiol. 2000; 279: F3-F11Google Scholar,24.Humes H.D. Cieslinski D.A. Coimbra T.M. Messana J.M. Galvao C. J. Clin. Invest. 1989; 84: 1757-1761Crossref PubMed Scopus (340) Google Scholar, 25.Coimbra T.M. Cieslinski D.A. Humes H.D. Am. J. Physiol. 1990; 259: F438-F443PubMed Google Scholar, 26.Norman J.T. Tsau Y.-K. Bacay A. Fine L.G. Clin. Sci. 1990; 78: 445-450Crossref PubMed Scopus (126) Google Scholar). These findings have led some investigators to propose protective and/or reparative roles for HB-EGF in the oxidant injury associated with renal ischemia-reperfusion (22.Hammerman M.R. Safirstein R. Harris R.C. Toback F.G. Humes H.D. Am. J. Physiol. 2000; 279: F3-F11Google Scholar, 23.Homma T. Sakai M. Cheng H.F. Yasuda T. Coffey R.J.J. Harris R.C. J. Clin. Invest. 1995; 96: 1018-1025Crossref PubMed Scopus (130) Google Scholar), so we directly tested this growth factor for the ability to both increase endogenous proximal tubule cell HK activity and mimic the protective effects of HK overexpression. In parallel, individual protective effects associated with either ectopic or endogenous HK activity were specifically examined for dependence upon the availability of Glc.EXPERIMENTAL PROCEDURESReagentsAll cell culture reagents, including serum and media additives, were supplied by Invitrogen (Grand Island, NY). Grade I yeast Glc 6-phosphate dehydrogenase and a lactate dehydrogenase (LDH) activity detection kit were purchased from Roche Molecular Biochemicals (Indianapolis, IN). The chemical fluorophores 4′,6′-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and YO-PRO-1 were obtained from Molecular Probes (Eugene, OR). All other reagents, including β-NADP, ATP, and recombinant human HB-EGF, were from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.Cell CultureMycoplasma-free HsPTC 2Originally designated HK-2 cells, this nomenclature is not used in the present work to avoid confusion with the HKII isoform. (Homos apiens ProximalTubule Cells) were obtained from the American Type Culture Collection (Rockville, MD) at passage 15. These cells are clonally derived from normal adult human proximal tubule cells that have been immortalized by transduction with human papillomavirus (HPV 16) E6 and E7 genes (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar). They exhibit both biochemical and morphological features of normal proximal tubule cells in culture (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar,27.Racusen L.C. Monteil C. Sgrignoli A. Lucskay M. Marouillat S. Rhim J.G.S. Morin J.-P. J. Lab. Clin. Med. 1997; 129: 318-329Abstract Full Text PDF PubMed Scopus (202) Google Scholar) and represent an established cell culture model of oxidant-induced proximal tubule injury (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar, 20.Zager R.A. Burkhart K. Kidney Int. 1997; 51: 728-738Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 21.Zager R.A. Burkhart K.M. Kidney Int. 1998; 53: 1661-1672Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Cells were routinely grown in monolayer culture on polystyrene dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ) or uncoated glass chamber slides (LabTek II, Nalge Nunc, Naperville, IL). Normal growth medium consisted of standard Dulbecco's modified Eagle:Ham's F-12 (1:1) medium supplemented with 5% fetal bovine serum as described previously (27.Racusen L.C. Monteil C. Sgrignoli A. Lucskay M. Marouillat S. Rhim J.G.S. Morin J.-P. J. Lab. Clin. Med. 1997; 129: 318-329Abstract Full Text PDF PubMed Scopus (202) Google Scholar) but with the following modifications: The medium was buffered with 20 mmHEPES, and the Glc content was reduced to 7.5 mm. Cells were maintained at 37 °C in a humidified 5% CO2incubator, and all experiments were performed between passages 15 and 30 to minimize the effects of phenotypic variation in continuous culture. Cells were also routinely serum-deprived for 16–24 h to induce quiescence prior to testing.Adenoviral Transgene Delivery and ExpressionRecombinant replication-deficient adenoviruses expressing HKI (rAd-HKI), the GLUT1 facilitative Glc transporter (rAd-GLUT1), and a β-galactosidase reporter gene (rAd-LacZ) (28.Becker T.C. Noel R.J. Coats W.S. Gómez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar) were obtained from Dr. Christopher B. Newgard (University of Texas Southwestern Medical Center, Dallas, TX) and were used to transfect quiescent HsPTC monolayers as described previously (29.Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (756) Google Scholar). Ectopic HKI expression was monitored by coupled enzymatic HK assays (see below), and β-galactosidase expression in paired LacZ-transfected control cells was monitored by a standard cytochemical method as detailed previously (30.Robey R.B. Osawa H. Printz R.L. Granner D.K. BioTechniques. 1996; 20: 40-42Crossref PubMed Scopus (12) Google Scholar). Susceptibility to oxidant-induced cell death was routinely examined 24 h following transfection, when transgene expression was maximal.Hexokinase AssaysWhole cell lysates were prepared by brief sonication (30–60 J) in ice-cold homogenization buffer (45 mm Tris-HCl, 50 mmKH2PO4, 10 mm Glc, 11.1 mm monothioglycerol, 0.5 mm EDTA, 0.2% (v/v) Triton X-100, pH 8.2), and HK activity was measured as the total Glc-phosphorylating capacity of fresh lysates using a standard Glc 6-phosphate dehydrogenase-coupled assay as described previously (31.Robey R.B. Ma J. Santos A.V.P. Am. J. Physiol. 1999; 277: F742-F749PubMed Google Scholar,32.Robey R.B. Raval B.J. Ma J. Santos A.V.P. Kidney Int. 2000; 57: 2308-2318Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) with minor modifications. The final assay mixture consisted of 1 unit/ml Glc 6-phosphate dehydrogenase, 0.5 mg/ml β-NADP, 6.7 mm ATP, 7 mm MgCl2, 4 mm Glc, 2.5 mm KH2PO4, 1 mm NaH2PO4, 11.1 mmmonothioglycerol, 0.01% (v/v) Triton X-100, 25 μm EDTA, and 45 mm Tris-HCl, pH 8.5. Total cellular protein content was assessed by the method of Bradford (33.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213220) Google Scholar) using bovine γ-globulin (Bio-Rad) as a reference standard, and all data were expressed as specific HK activity in units per g of total protein, where 1 unit is defined as that amount of enzyme activity resulting in the coupled formation of 1 μmol of NADPH per min at 25 °C.Metabolic AssaysGlc utilization and lactate production were evaluated as described previously (31.Robey R.B. Ma J. Santos A.V.P. Am. J. Physiol. 1999; 277: F742-F749PubMed Google Scholar, 32.Robey R.B. Raval B.J. Ma J. Santos A.V.P. Kidney Int. 2000; 57: 2308-2318Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and were assayed as the net rates of Glc disappearance and lactate accumulation in the culture medium, respectively. For these experiments, cells were tested in normal growth medium containing 7.5 mm Glc and lacking both serum and phenol red. Where appropriate, medium aliquots were assayed colorimetrically for both Glc and lactate content. Net cellular reductive capacity was also monitored using a commercially available tetrazolium reduction assay (CellTiter 96 AQueousNon-Radioactive Cell Proliferation Assay, Promega; Madison, WI). This in situ assay, which relies on the ability of viable cells to reduce MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (34.Barltrop J.A. Owen T.C. Cory A.H. Cory J.G. Bioorg. Med. Chem. Lett. 1991; 1: 611-614Crossref Scopus (367) Google Scholar)) in the presence of the electron acceptor phenazine ethosulfate, is widely employed to monitor net cellular reductive capacity as an indicator of both proliferation and viability. Originally attributed to mitochondrial metabolism (35.Slater T.F. Sawyer B. Sträuli U. Biochim. Biophys. Acta. 1963; 77: 383-393Crossref PubMed Scopus (633) Google Scholar), it has subsequently been demonstrated that tetrazolium bioreduction to yield formazan chromophores is primarily extramitochondrial and largely involves the pyridine dinucleotides NADH and NADPH (36.Berridge M.V. Tan A.S. Arch. Biochem. Biophys. 1993; 303: 474-482Crossref PubMed Scopus (1088) Google Scholar). It has also been shown that formazan generation is critically dependent upon both the uptake and continued metabolism of Glc (37.Vistica D.T. Skehan P. Scudiero D. Monks A. Pittman A. Boyd M.R. Cancer Res. 1991; 51: 2515-2520PubMed Google Scholar, 38.Segu V.B.G. Li G. Metz S.A. Metabolism. 1998; 47: 824-830Abstract Full Text PDF PubMed Scopus (27) Google Scholar), presumably via the coupled reduction of pyridine dinucleotides. Thus, chromophore formation will necessarily reflect both ongoing metabolic activity and oxidative consumption of reduced nicotinamide cofactor pools. MTS reduction assays were performed according to the manufacturer's recommendations and were exploited to monitor net cellular reductive capacity in real-time as described previously (38.Segu V.B.G. Li G. Metz S.A. Metabolism. 1998; 47: 824-830Abstract Full Text PDF PubMed Scopus (27) Google Scholar).Acute Oxidative Stress ModelThe oxidative stress model used for these studies was essentially as described by Zager and colleagues (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar, 20.Zager R.A. Burkhart K. Kidney Int. 1997; 51: 728-738Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 21.Zager R.A. Burkhart K.M. Kidney Int. 1998; 53: 1661-1672Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) with minor modifications. HsPTC cells were routinely grown to ∼90% confluence in normal growth medium before serum deprivation overnight to induce quiescence. In preliminary testing, serum deprivation alone, for periods up to 24 h, was not sufficient to induce apoptosis in these cells. 3J. M. Bryson and R. B. Robey, unpublished observations. Quiescent cell monolayers were then exposed to H2O2 at concentrations up to 10 mm for as long as 3 h at 37 °C in Hanks' balanced salt solution (HBSS) containing physiologic levels of Glc (5.6 mm).Cell Viability/Death AssaysIn initial experiments, cell death was monitored microscopically by the uptake of both YO-PRO-1 (39.Idziorek T. De Estaquier J. Bels F. Ameisen J.C. J. Immunol. Meth. 1995; 185: 249-258Crossref PubMed Scopus (335) Google Scholar) and PI, fluorescent markers of apoptosis and necrosis, respectively. Quiescent HsPTC monolayers were exposed to H2O2 (0–10 mm) for up to 3 h at 37 °C in HBSS containing 1 μm YO-PRO-1 and/or 1.5 μm PI. The cellular uptake of both fluorophores was monitored, alone and in combination, using a Carl Zeiss LSM 410 laser scanning confocal microscope system equipped with an argon/krypton laser as previously described (40.Robey R.B. Ruiz O. Santos A.V.P. Ma J. Kear F. Wang L.-J. Li C.-J. Bernardo A.A. Arruda J.A.L. Biochemistry. 1998; 37: 9894-9901Crossref PubMed Scopus (74) Google Scholar), and unstressed control cells were routinely analyzed in parallel to exclude direct toxic effects of the fluorophores. The exclusion of both YO-PRO-1 and PI from viable, non-apoptotic cells makes them useful to screen for cell death in real-time, but because not all cells are visualized by this method, additional measures are required for quantitative estimates of cell death. For this reason, quantitative assessment of apoptosis was performed under selected conditions by conventional DAPI staining and cell counting (29.Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (756) Google Scholar, 41.Kennedy S.G. Wagner A.J. Conzen S.D. Jordán J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (977) Google Scholar). In these experiments, cells were fixed, where appropriate, by the direct addition of formaldehyde to the medium to a final concentration of 10% (v/v). Detached cells were allowed to settle and adhere to the slide before DAPI staining and analysis using a Zeiss Axioplan 2 fluorescence microscopy system equipped with a matched ultraviolet light source. Apoptotic cells were manually counted and expressed as a percentage of the total cell number. At least four independent fields of ∼100 cells were scored for each experimental condition.Cytolysis AssaysPlasma membrane integrity was serially assessed by monitoring cytosolic lactate dehydrogenase (LDH) release into the culture medium using a commercially available assay kit (Roche Molecular Biochemicals) per the manufacturer's recommendations. In brief, LDH activity was colorimetrically assayed in cell-free aliquots of culture supernatants via the coupled enzymatic reduction of a tetrazolium salt to generate a formazan chromophore. The total releasable pool of cellular LDH activity was always assayed in identical paired cell monolayers following lysis with 1% (v/v) Triton X-100, and the proportion of the total activity in the supernatant was taken as an index of the degree of cell lysis.Statistical AnalysisAll data are presented as the means ± S.E. for at least three independent experiments. Statistical comparisons were performed by two-tailed paired t-testing using a significance level of 95% and StatView 5.0.1 software for Macintosh computers (SAS Institute, Cary, NC).RESULTSH2O2 Decreases Both HsPTC Viability and Net Cellular Reductive Capacity in a Time- and Concentration-dependent MannerTo characterize the general response of HsPTC to acute oxidative stress, quiescent cell monolayers were exposed to varying concentrations of H2O2 (0–10 mm) in the presence of fluorescent indicators of apoptosis (1 μm YO-PRO-1) and/or necrosis (1.5 μm PI). H2O2increased the cellular uptake of both fluorophores in a concentration- and time-dependent manner, with virtually all cells exhibiting YO-PRO-1 uptake and nuclear condensation characteristic of apoptosis within 2 h of exposure to 10 mmH2O2 (Fig. 1A). PI uptake exhibited similar H2O2 concentration dependence but typically lagged behind YO-PRO-1 uptake by 0.5–1.0 h. 3J. M. Bryson and R. B. Robey, unpublished observations. In addition to this temporal delay, PI uptake by cells incubated with both fluorophores was observed only in YO-PRO-1-staining apoptotic cells, suggesting the development of secondary necrotic changes in these cells. Acute oxidative stress was also associated with marked depression of in situ tetrazolium bioreduction (Fig. 1B). This effect was similarly concentration-dependent and was most marked at H2O2 concentrations associated with increased early apoptosis (Fig. 1A). Changes in net cellular reductive capacity, however, uniformly preceded the uptake of both YO-PRO-1 and PI, which is temporally compatible with roles for both metabolism and cellular redox status in this model of acute oxidant-induced cell death.Adenoviral HKI Transgene Delivery Increases Total HK Activity in a Titer-dependent MannerTo directly assess the functional consequences of primary increases in cellular Glc phosphorylating capacity, HsPTC were transfected with recombinant adenoviruses expressing either a β-galactosidase reporter gene (rAd-LacZ) or an HKI transgene. As depicted in Fig. 2A, control adenoviral transfections using the β-galactosidase-expressing vector (rAd-LacZ) resulted in uniform, titer-dependent reporter gene expression. HKI overexpression using a matched vector (rAd-HKI; Fig. 2B) similarly increased total HK activity in a titer-dependent manner. Basal HK activity (17 ± 1 units/g of protein) in untransfected HsPTC was comparable in magnitude to that reported previously for both this cell line (27.Racusen L.C. Monteil C. Sgrignoli A. Lucskay M. Marouillat S. Rhim J.G.S. Morin J.-P. J. Lab. Clin. Med. 1997; 129: 318-329Abstract Full Text PDF PubMed Scopus (202) Google Scholar) and for primary cultures of human proximal tubule cells (42.Courjault-Gautier F. Chevalier J. Abbou C.C. Chopin D.K. Toutain J.H. J. Am. Soc. Nephrol. 1995; 5: 1949-1963Crossref PubMed Google Scholar). In paired cell monolayers, infection with rAd-HKI at a relative multiplicity of infection (m.o.i.) of ∼1 increased total HK activity at 24 h by over 40% (25 ± 2 units/g of protein, p < 0.001), whereas a 10-fold higher viral titer increased total HK activity nearly 4-fold (64 ± 7 units/g of protein, p < 0.001).Figure 2Adenoviral HKI transgene delivery increases total HK activity in a concentration-dependent manner. Confluent cell monolayers were routinely transfected with matched recombinant pJM17-based adenoviruses (28.Becker T.C. Noel R.J. Coats W.S. Gómez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar) expressing a β-galactosidase reporter gene (rAd-LacZ) or a rat HKI transgene (rAd-HKI). As shown in A, transfections with rAd-LacZ resulted in uniform, titer-dependent reporter gene expression, with virtually all cells expressing cytochemically detectable β-galactosidase at viral multiplicities-of-infection (m.o.i.) ≥ 1. A representative set of control transfections is shown. As depicted in B, transfection with rAd-HKI resulted in similar titer-dependent HKI transgene expression. At an m.o.i. ∼ 1, rAd-HKI increased total HK activity by over 40% at 24 h (*, p < 0.001). Exposure to a 10-fold greater titer of rAd-HKI (m.o.i. ∼ 10) resulted in a corresponding 4-fold increase in total HK activity (*, p < 0.001). Higher viral titers were associated with much smaller increments in HK activity and were variably accompanied by evidence of cellular toxicity, ostensibly due to higher levels of native adenoviral gene product expression (29.Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (756) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cells Expressing Ectopic HKI Exhibit Decreased Susceptibility to Acute Oxidant-induced Cell Deathβ-Galactosidase-expressing (+rAd-LacZ), recombinant HKI-expressing (+rAdHKI), and paired untransfected HsPTC cell monolayers were acutely exposed to H2O2over a concentration range (1–10 mm) previously required to induce rapid oxidant-induced death in both these cells (19.Ryan M.J. Johnson G. Kirk J. Fuerstenberg S.M. Zager R.A. Torok-Storb B. Kidney Int. 1994; 45: 48-57Abstract Full Text PDF PubMed Scopus (713) Google Scholar, 20.Zager R.A. Burkhart K. Kidney Int. 1997; 51: 728-738Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 21.Zager R.A. Burkhart K.M. Kidney Int. 1998; 53: 1661-1672Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and isolated proximal tubule segments (21.Zager R.A. Burkhart K.M. Kidney Int. 1998; 53: 1661-1672Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 43.Zager R.A. J. Clin. Invest. 1992; 90: 711-719Crossref PubMed Scopus (123) Google Scholar). As shown in Fig. 3, cells overexpressing HKI (+rAd-HKI, m.o.i. ∼1; lower panels) exhibited improved cell morphology and attachment, as well as decreased uptake of both YO-PRO-1 and PI, relative to paired untransfected controls (wt; upper panels) or cells expressing a β-galactosidase reporter gene (+rAd-LacZ, m.o.i. ∼1; middle panels) when exposed to 10 mm H2O2 for 3 h. Qualita
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