Oxidative Stress Mediates Apoptotic Changes Induced by Hyperglycemia in Human Tubular Kidney Cells
Daniela Verzola*,
Maria Bianca Bertolotto,
Barbara Villaggio*,
Luciano Ottonello,
Franco Dallegri,
Francesca Salvatore*,
Valeria Berruti*,
Maria Teresa Gandolfo*,
Giacomo Garibotto* and
Giacomo Deferrari*
*Division of Nephrology, Department of Internal Medicine, University of Genoa, Genoa, Italy; and Division of Internal Medicine Department of Internal Medicine, University of Genoa, Genoa, Italy
Correspondence to Dr. Giacomo Garibotto, Department of Internal Medicine, Nephrology Division, Viale Benedetto XV 6, 16132 Genoa, Italy. Phone: ++390103538989; Fax: ++390103538959; E-mail: gari{at}unige.it
ABSTRACT. Reactive oxygen species (ROS) are important mediatorsfor several biologic responses, including apoptosis. The presentstudy evaluated the time course of changes in intracellularROS production and apoptosis-related proteins, as well as apoptoticchanges in human tubular proximal cells (HK-2 cells) exposedto hyperglycemia. Apoptosis (annexin V binding), ROS formation(fluorescence probe dichlorofluorescin diacetate and FACScanflow cytometry), and X chromosome–linked protein (XIAP;Western blot) were studied in HK-2 cells grown in a medium containingnormal (NG) or high glucose (HG) concentrations (5.5 or 30 mM,respectively) for 18 to 48 h. HG promoted an increase (65% at18 h and 73% at 24 h; P < 0.05 versus NG) in intracellularROS generation. At 18 h, the NF-kB binding activity (evaluatedby electrophoretic mobility-shift assay) was suppressed by HG.At the same time, the expression of NF-kB–induced antiapoptoticXIAP was reduced in HG-treated cells. Apoptotic changes wereobserved at 48 h (34 ± 7% in HG versus 10 ± 3%in NG; P < 0.001). Changes in ROS production at 24 h predictedchanges in the apoptotic index at 48 h (r = 0.96, P < 0.0001).These results suggest that hyperglycemia induces apoptotic changesin human tubular cells via an increase in oxidative stress andthat a downregulation of antiapoptotic protein XIAP is a componentof this response.
Most studies on diabetic nephropathy have focused on functionaland structural changes in the glomeruli. However, alterationsin tubular function have been observed as an early phenomenonin diabetes (1). Moreover, changes within the tubulointerstitiumoccur in a large subset of patients with diabetic nephropathyand play a major role in determining the renal outcome (2).The mechanisms by which diabetes leads to tissue damage andcontributes to the development of tubulointerstitial damageare not understood. Recent observations indicate that hyperglycemiatriggers the generation of free radicals and oxidant stress(3) in both mesangial (4) and tubular cells (5,6). Reactiveoxygen species (ROS) are considered to be important mediatorsfor several biologic responses, including proliferation, extracellularmatrix deposition, and apoptosis (7). Although in the past ROShave been regarded as toxic metabolites, this concept has beenrevised in consideration of recently proposed roles for ROSas second messenger (4). It has been suggested that cell-typespecificity does occur as for signaling events by which cellsrespond to ROS, thus explaining the different response in differentcell types (4).
Apoptosis might contribute to the structural changes that occurin the diabetic kidney (8). We have previously observed thathigh glucose (HG) early induces apoptotic changes in tubularcells and that these changes are prevented by several endogenousand exogenous antioxidants (5). To elucidate further the possiblerelations between oxidative stress and apoptosis in tubularcells, we studied the time course of changes in intracellularROS production and apoptosis-related proteins, as well as apoptoticchanges in human tubular proximal cells (HK-2 cell line) exposedto hyperglycemia. Our data support the hypothesis that apoptosisinduced by hyperglycemia is mediated by ROS and that a downregulationof antiapoptotic X chromosome–linked protein (XIAP) isa major component of this response.
HK-2 cells, an immortalized proximal tubular epithelial cell(PTEC) line from normal adult human kidney, were obtained fromAmerican Type Culture Collection. Cells were grown to subconfluenceand maintained for 18 to 48 h in serum-free medium containing5.5 (NG) or 30 (HG) mM glucose. In separate experiments, HK-2were maintained in serum-free medium containing 5.5 mM glucose+ 24.5 mM mannitol (HM) (5). To evaluate apoptotic phenomena,we considered the percentage of cells that were annexin V positive/propidiumiodide negative (5). For Western blot analysis, cell pelletswere treated as described previously (5). Briefly, equal amountsof proteins (40 µg/lane) were diluted with nonreducingsample buffer and separated by electrophoresis on a 12% SDS-PAGEgel. The proteins were electrotransferred to Hybond-C-nitrocellulosemembrane. For immunoblot analysis, blots were incubated withprimary antibody (anti-XIAP) for 1 h at room temperature onshaking, then washed in PBS/Tween 20 0.05% (vol/vol), pH 7.4,and, finally, incubated in secondary horseradish peroxidase–labeledantibody for 1 h at room temperature. Bound secondary antibodywas detected using enhance chemiluminescence. The intracellularformation of ROS was detected using the fluorescence probe 2'-7'dichlorofluorescin diacetate. After treatment, HK-2 were treatedas described previously (9). To evaluate electrophoretic mobility-shiftassay (EMSA) for NF-kB activity, nuclear extracts were preparedfrom HK-2 cells by using the NE-PER Nuclear and CytoplasmaticExtraction Kit (Pierce/Celbio, Pero, Italy) according to themanufacturer’s instruction. EMSA was performed using thekB DNA sequence of the Ig gene (5'-CCGGTCAGGGGACTTTC-CGAGACT).Nuclear extracts (2 µg) were incubated with 50 kcpm of32P-labeled NF-kB oligonucleotide in a binding reaction mixture(10 mM Tris-HCl [pH 7.5], 80 mM NaCl, 1 mM EDTA, 1 mM DTT, 5%glycerol, and 1.5 µg of poly [dI-dC]) for 30 min in ice.The DNA-protein complexes were separated on 6% polyacrylamidegels.
All data are presented as means ± SD. Significant differenceamong data sets was determined by either the unpaired t testor ANOVA with post hoc testing (Statview; Abacus, Berkeley,CA). Differences were considered to be statistically significantat P < 0.05.
Time Course of Intracellular ROS Production and Apoptotic Changes Induced by HG in PTEC
The exposure of HK-2 cells to 30 mM glucose was associated withan early increase in ROS production, which declined thereafter(Figure 1A). ROS production was already increased (by approximately65%) at 18 h and augmented by 75% after 24 h (P < 0.05 versusNG). At 48 h, ROS production was lower than baseline values.Changes in ROS production at 24 h predicted apoptosis at 48h (r = 0.96, P < 0.0001). Mannitol 30 mM had no effect onROS formation.
Figure 1. Effect of glucose (5.5 or 30 mmol/L) on intracellular generation of reactive oxygen species (ROS; A) and apoptosis assessed by annexin V/propidium iodide (B). (A) For studying ROS, cells were treated with normal glucose (NG), high glucose (HG), or high mannitol (HM) for 18, 24, and 48 h and then treated with 5 µmol/L dichlorofluorescin diacetate for 5 min (*P < 0.05 versus NG). (B) Effects of NG, HG, and M on apoptosis. Proximal tubular epithelial cells were treated for 24 to 48 h. (*P < 0.001 versus NG). Data are expressed as means ± SD and are representative of four separate experiments. Every experiment was carried out in duplicate.
Exposure of HK-2 cells to 30 mM glucose for 24 h did not resultin significant changes in apoptosis. When cells were subjectedto HG for 48 h, we observed a significant appearance in earlyapoptotic changes (34 ± 7% of annexin-positive, propidium-negativecells versus 10 ± 3% for 5.5 mM glucose; P < 0.001;Figure 1B). A similar change in osmolarity obtained by the useof 30 mM mannitol failed to induce apoptotic changes.
Effects of HG on Apoptosis-Related Proteins
HG-mediated apoptosis was preceded at 18 h by a decrease inthe expression of antiapoptotic protein XIAP (Figure 2). Thisevent was not observed in HK-2 grown in normal glucose. HG-mediatedapoptosis was associated with an increase in the Bax/Bcl2 ratioat 48 h (data not shown; immunostaining and Western Blot analysis).Again, these effects were not reproduced by high osmolality.NF-kB binding activity was markedly reduced when cells weregrown for 18 h in HG (Figure 2). The effect of HG was not reproducedby normal glucose.
Figure 2. (A) Expression of X chromosome–linked protein (XIAP) was evaluated by Western blot. Cells subjected to HG for 18 h showed the absence of XIAP protein with respect to NG. (B) NF-kB binding to DNA under NG and HG condition in HK-2 cells at 18 h. NF-kB–binding activity is blunted in HG-treated cells.
We have observed that the increase in intracellular ROS productionduring incubation of PTEC with HG media is an early effect,which precedes by several hours the occurrence of apoptoticchanges. Lately, when a significant percentage of cells showevidence of apoptosis, intracellular ROS formation declines.According to these findings, ROS generated by accelerated glucosemetabolism might represent a signaling molecule for early apoptoticchanges. Are the increase in intracellular ROS production andapoptosis related phenomena, or do they represent the concurrenceof unrelated effects? The prevention of apoptosis in PTEC bydifferent antioxidants, such as GSH, N-acetylcysteine (NAC),and taurine, as shown recently (5), indicates a cause–effectrelationship between ROS and early apoptotic changes. In addition,we observed that the early changes in intracellular ROS formationinduced by HG predicted the variations in the apoptotic index.This suggests a close relationship between ROS formation andapoptosis. Moreover, concurrent with the increased ROS formation,the expression of XIAP was blunted by HG medium. XIAP is partof the family of cellular inhibitors of apoptosis, which directlybind and inhibit effector caspases, such as caspase-3 and -7,as well as prevent activation of pro-caspase-6 and -9 (10).XIAP can inhibit caspases by preventing cleavage by pro-caspasesor by direct inhibition of activated caspases (10). Accordingto these findings, hyperglycemia may exert a permissive effectof apoptosis of PTEC by downregulating the endogenous inhibitorsof caspases.
It is suggested that cellular inhibitors of apoptosis are regulatedby NF-kB activity (11). In several cell lines, NF-kB has beenidentified as a target for ROS-dependent signals. Studies aboutthe influence of NF-kB expression on cell survival after oxidantshave resulted in conflicting results. In some studies, NF-kBhas been suggested to cause a protective effect in responseto oxidative stress (12), whereas others support a role of NF-kBas a proapoptotic factor (13). To determine whether NF-kB isinfluenced by ROS-dependent signals in HK-2 maintained in 30mM glucose, we performed gel shift assays with nuclear proteinsand an NF-kB binding site–specific probe. We observedthat when PTEC were grown for 18 h in HG, NF-kB binding activitywas switched off. This finding, in addition to those showinga decrease in the expression of NF-kB–induced XIAP, suggeststhat in this experimental setting, NF-kB has a prosurvival andantiapoptotic function.
In summary, data presented here suggest that ROS are generatedas an early signal in human PTEC, which subsequently developapoptotic changes under HG media, implicating ROS as potentialmediators of glucose-induced apoptosis. A downregulation ofantiapoptotic protein XIAP seems to be a major component ofthis response. These findings are consistent with several otherobservations implicating ROS in the pathogenesis of diabeticcomplications.
Acknowledgments
This study was supported by grants from the University of Genoaand MIUR (FIRB project).
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Abstract
Introduction
