Inhibition of apoptosis by Zn 2+ in renal tubular cells following ATP depletion

【摘要】  Apoptosis has been implicated in ischemic renal injury. Thus one strategy of renal protection is to antagonize apoptosis. However, apoptosis inhibitory approaches remain to be fully explored. Zn 2+ has long been implicated in apoptosis inhibition; but systematic analysis of the inhibitory effects of Zn 2+ is lacking. Moreover, whether Zn 2+ blocks renal cell apoptosis following ischemia is unknown. Here, we demonstrate that Zn 2+ is a potent apoptosis inhibitor in an in vitro model of renal cell ischemia. ATP depletion induced apoptosis in cultured renal tubular cells, which was accompanied by caspase activation. Zn 2+ at 10 µM inhibited both apoptosis and caspase activation, whereas Co 2+ was without effect. In ATP-depleted cells, Zn 2+ partially prevented Bax activation and cytochrome c release from mitochondria. In isolated cell cytosol, Zn 2+ blocked cytochrome c -stimulated caspase activation at low-micromolar concentrations. In addition, Zn 2+ could directly antagonize the enzymatic activity of purified recombinant caspases. We conclude that Zn 2+ is a potent inhibitor of apoptosis in renal tubular cells following ATP depletion. Zn 2+ blocks apoptosis at multiple steps including Bax activation, cytochrome c release, apoptosome function, and caspase activation.

【关键词】  ischemia renal tubule

A BROAD RANGE OF CLINICAL conditions including dehydration, hypotension, septic shock, trauma, and operative arterial clamping leads to the reduction of blood flow to the kidneys. When blood supply to the kidneys is inadequate to sustain cellular demands, cell injury and tissue damage ensue, resulting in ischemic acute renal failure (ARF) ( 6 ). Despite intensive care and hemodialysis, severe ischemic ARF continues to be associated with high mortality. The development of ischemic ARF involves multiple factors and may proceed in several phases ( 6, 11, 17, 33, 34, 36, 38, 44 ). A major pathological feature of ischemic ARF is sublethal and lethal damage to renal tubules ( 28 ). Under these conditions, tubular cell death in the necrotic form can be identified, and as a result, the syndrome is termed "acute tubular necrosis." On the other hand, compelling evidence has now suggested a role for apoptosis in ischemic ARF ( 5, 38, 41, 49 ). Morphologically, apoptotic cells were identified in ischemia-reperfused kidneys ( 24, 42 ). Biochemically, renal ischemia-reperfusion led to the expression and activation of caspases, a family of cysteine proteases responsible for disassembly of apoptotic cells ( 23 ). Endonuclease activation was also documented ( 4 ). In addition, regulation of apoptotic genes including the Bcl-2 family has been shown in kidneys following ischemia ( 3, 20 ). Finally, several pharmacological agents appeared to ameliorate ischemic renal injury, at least in part, by diminishing apoptosis ( 9, 24, 25 ). In this regard, our recent work suggests that minocycline, a tetracycline derivative, may upregulate Bcl-2 to attenuate apoptosis during renal ischemia-reperfusion and preserve renal function ( 51 ).

Mechanistically, tubular cell apoptosis during ischemia-reperfusion may follow two major pathways. In the extrinsic pathway, ligand binding of death receptors leads to oligomerization of the receptor protein and subsequent formation of death-inducing signaling complexes and the activation of caspase-8 ( 2 ). In the intrinsic pathway, cellular stress leads to mitochondrial disruption, releasing apoptogenic molecules such as cytochrome c ( 7 ). Cytochrome c then associates with Apaf-1 in the cytosol, resulting in the recruitment and activation of caspase-9. Using in vitro models, we documented the release of cytochrome c from cultured tubular cells during hypoxic-ischemic incubation ( 40 ). The cells with released cytochrome c activate caspases and exhibit typical apoptotic morphology including cellular shrinkage, nuclear condensation, and fragmentation ( 12, 40 ). Importantly, our subsequent studies identified a critical role for Bax, a proapoptotic Bcl-2 family protein, in mitochondrial damage and cytochrome c release ( 16, 32, 40 ). During ischemic incubation, Bax translocates from the cytosol to mitochondria, where the molecules insert into the outer membranes, oligomerize, and presumably form pathological pores on the mitochondrial membranes, releasing cytochrome c. Expression of Bcl-2 antagonizes Bax in mitochondria and prevents cytochrome c leakage, which is accompanied by the inhibition of apoptosis and long-term cell survival ( 32, 40 ). These observations have been confirmed and extended in related models of tubular cell apoptosis induced by in vitro ATP depletion ( 10, 27 ) and in vivo ischemia-reperfusion of cadaveric kidney allografts ( 8 ). Our recent work further extended these observations by selecting death-resistant cells through repeated episodes of hypoxia ( 14 ). The selected cells overexpress Bcl-X L, which prevents Bax activation and subsequent cytochrome c release, resulting in the preservation of cell viability ( 14 ).

The role played by apoptosis in tubular cell injury suggests that it is possible to reduce tissue damage and ameliorate ischemic renal failure by antagonizing apoptosis ( 5, 38, 41, 49 ). Indeed, caspase inhibitors have been used to diminish ischemic renal failure in vivo in animal models ( 9, 31 ). However, despite our molecular understanding of apoptosis, approaches of apoptosis inhibition remain very limited. Zn 2+ has long been implicated in apoptosis inhibition ( 48 ). Originally, Zn 2+ was shown to be an inhibitor of endonucleases, and as a result, preventing internucleosomal DNA degradation during apoptosis ( 18 ). Later studies indicated that Zn 2+ was able to inhibit caspases ( 43, 45 ). Recently, Zn 2+ was shown to suppress apoptosis at the mitochondrial level, blocking Bax translocation and cytochrome c release ( 19 ). Despite these observations, systematic analysis of the inhibitory effects of Zn 2+ was lacking. The current study was designed to 1 ) determine whether Zn 2+ can inhibit tubular cell apoptosis following ischemic incubation or ATP depletion and 2 ) identify the apoptotic steps that are suppressed by Zn 2+.

MATERIALS AND METHODS

Materials. Rat kidney proximal tubular epithelial cells were originally obtained from Dr. U. Hopfer at Case Western Reserve University (Cleveland, OH). The cells were maintained and plated for experiments as described previously ( 54 ). Free 7-amino-4-trifluoromethylcoumarin (AFC) and DEVD.AFC were purchased from Enzyme Systems Products (Dublin, CA). Active recombinant caspase-3 was from Gene Therapy Systems (San Diego, CA). The rabbit polyclonal antibody specific to the active form of caspase-3 was a gift from Dr. A. Srinivasan at Idun Pharmaceuticals (La Jolla, CA). Other antibodies were purchased from the following sources: monoclonal anti-cyt.c (7H8.2C12 and 6H2.B4) from BD Pharmingen (San Diego, CA), monoclonal anti-Bax (1D1) from NeoMarkers (Fremont, CA), and all secondary antibodies from Jackson ImmunoResearch (West Grove, PA). Other reagents were from Sigma (St. Louis, MO).

ATP depletion. ATP depletion of cultured tubular cells was conducted as detailed recently ( 53 ). Briefly, cells were incubated with 10 mM azide for 3 h in glucose-free Krebs-Ringer bicarbonate solution (composition in mM: 115 NaCl, 3.5 KCl, 25 NaHCO 3, 1 KH 2 PO 4, 1.25 CaCl 2, and 1 MgSO 4; gassed with 5% CO 2 ). After ATP depletion, groups of cells were returned back to full culture medium to simulate in vivo reperfusion.

Morphological examination of apoptosis. Apoptosis was assessed by morphological methods as described in our previous publications ( 13 - 16 ). Cells were exposed to 5 µg/ml of Hoechst 33342 for 2-5 min in PBS to stain the nucleus. Cellular morphology was then monitored by phase contrast microscopy. Nucleus stained with Hoechst 33342 was examined by fluorescence microscopy. Typical apoptotic morphology included cellular shrinkage, nuclear condensation and fragmentation, and formation of apoptotic bodies.

Measurement of caspase activity. The enzymatic activity of caspases was measured by a method modified from our previous work, using the fluorogenic peptide substrate DEVD.AFC ( 12 - 14 ). Briefly, cells were extracted with 1% Triton X-100. The resultant lysates of 25 µg protein were added to enzymatic reactions containing 50 µM DEVD.AFC. After 60 min of reaction at 37°C, fluorescence at excitation 360 nm/emission 530 nm was monitored by a GENios plate-reader (Tecan US, Research Triangle Park, NC). For each measurement, a standard curve was constructed using free AFC. Based on the standard curve, the fluorescence reading from each enzymatic reaction was translated into the nanomolar amount of liberated AFC to indicate caspase activity.

Immunofluorescence of cytochrome c, Bax, and active caspase-3. Indirect immunofluorescence was conducted as described in our previous work ( 14, 16, 51, 53 ). Cells were grown on the collagen-coated glass coverslip. For active caspase-3 or Bax, the cells were fixed with 4% paraformaldehyde, blocked with 2% normal goat serum, exposed to a rabbit polyclonal antibody specific for active caspase-3 or a mouse monoclonal anti-Bax, and finally incubated with a Cy3-labeled goat anti-rabbit or a goat anti-mouse secondary antibody. For immunofluorescence of cytochrome c, cells were fixed with a modified Zamboni's fixative containing picric acid and 4% paraformaldehyde. After being blocked in 2% normal goat serum, the cells were incubated with a mouse monoclonal anti-cytochrome c, followed by exposure to Cy3-labeled goat anti-mouse secondary antibody. Signals were examined by fluorescence microscopy using Cy3 channel.

Cellular fractionation. To analyze Bax translocation to mitochondria and cytochrome c release from the organelles, cells were fractionated into cytosolic fraction and the membrane-bound organellar fraction, which was enriched with mitochondria. The fractionation was achieved by using low concentrations of digitonin, as described in our previous work ( 14, 16, 40, 51 ). Digitonin at low concentrations selectively permeabilizes the plasma membranes, without solubilizing mitochondria. This method has also been verified and used by other investigators examining protein translocations during apoptosis ( 35, 39, 52 ). Briefly, cells were incubated with 0.05% digitonin in isotonic sucrose buffer (in mM: 250 sucrose, 10 HEPES, 10 KCl, 1.5 MgCl 2, 1 EDTA, and 1 EGTA; pH 7.1) for 2 min at room temperature. The released cytosol was collected. Digitonin-insoluble part was further extracted with 2% SDS buffer to collect the membrane-bound organellar fraction. Because Bax and cytochrome c redistribution during apoptosis mainly takes place between the cytosol and mitochondria, immunoblot analysis of the organellar fraction is expected to reveal mitochondrial content of the molecules.

Immunoblot analysis. Immunoblot analysis of proteins was performed in a NuPAGE Gel System. After electrophoresis, the proteins were electroblotted onto PVDF membranes. The blots were blocked with 2% BSA and then exposed to the primary antibodies overnight at 4°C. Finally, the blots were incubated with the horseradish-peroxidase-conjugated secondary antibody, and antigens on the blots were revealed using the enhanced chemiluminescence kit (Pierce, Rockford, IL).

In vitro reconstitution of caspase activation. Reconstitution of caspase activation by adding exogenous cytochrome c to isolated cytosol was conducted by a method modified from our previous work ( 12, 14, 16, 51 ). A major modification was that no EGTA or EDTA was present in the reconstitution mixture to avoid Zn 2+ chelation. Briefly, cytosol was extracted from control cells with 0.05% digitonin, concentrated to 4-5 mg protein/ml with 3-kDa cutoff microconcentrators, and kept frozen at -70°C. For reconstitution, 1 µl of 0.5 mg/ml cytochrome c and 1 µl of 10 mM dATP were added to 7.5 µl of cytosolic extracts containing 25 µg of protein and incubated for 1 h at 30°C. After incubation, the reconstitution mixture was transferred to a buffer containing 50 µM DEVD.AFC to determine caspase activity. To test the effects of Zn 2+, the ions were added at indicated concentrations during the reconstitution.

Recombinant caspase-3 assay. The enzymatic activity of purified recombinant caspase-3 was measured as described previously ( 15 ), with two modifications to avoid Zn 2+ chelation. First, 10 mM DTT in the reaction buffer was replaced by -mecaptoethanol. Second, EGTA and EDTA were omitted from the reaction. A half unit of caspase-3 was added to the reaction buffer containing DEVD.AFC in the absence or presence of indicated amounts of Zn 2+. Fluorescence of liberated AFC was measured every 10 min during the reaction. The signal was converted to molar amounts of AFC based on the standard curve.

Statistics. Data are expressed as means ± SD ( n 3). Statistical differences between two groups were determined using Student's t -test with Microsoft EXCEL 2002. P < 0.05 was considered to reflect significant differences.

RESULTS

Inhibition of ATP depletion induced apoptosis by Zn 2+. ATP depletion is a primary cause of cell injury and tissue damage following renal ischemia-reperfusion in vivo. As a result, common in vitro models to study ischemic renal injury involve the depletion of cell ATP ( 29 ). This can be achieved by blocking ATP production from mitochondrial respiration and limiting anaerobic glycolysis. In our previous work, ATP depletion was induced in cultured renal tubular cells by severe hypoxia or mitochondrial inhibition in glucose-free medium ( 12, 13, 15, 40, 51, 53 ). Under these conditions, the mitochondrial pathway of apoptosis was activated, resulting in cytochrome c release and caspase activation. When the cells were subsequently returned to full culture medium for "reperfusion," typical morphology of apoptosis developed. The current study used this well-characterized apoptotic model to examine the effects of Zn 2+. As shown in Fig. 1, cultured renal tubular cells underwent apoptosis after ATP depletion by azide, showing cellular and nuclear shrinkage, condensation, and fragmentation. Zn 2+, when added during ATP depletion and subsequent reperfusion, blocked apoptosis at micromolar concentrations. The inhibitory effects of Zn 2+ appeared specific, because the control divalent ion Co 2+ was without effect. To quantify apoptosis, we counted the cells that developed typical apoptotic morphology ( Fig. 1 B ). The basal level of apoptosis in control was 2.7%. After 3 h of ATP depletion and 1 h of reperfusion, 37.7% of cells became apoptotic. Zn 2+ at 10 µM suppressed apoptosis to 12.2%, whereas the Co 2+ group had 34.2% apoptosis.

Fig. 1. Zn 2+ inhibition of tubular cell apoptosis following ATP depletion. Cultured renal tubular cells were incubated with 10 mM azide for 3 h in glucose-free medium and then returned to full culture medium for 2 h of reperfusion. For the conditions with Zn 2+ or Co 2+, the ions were present during azide incubation and reperfusion. The cells were fixed and stained with Hoechst 33342. A : phase contrast images and Hoechst staining of the same fields of cells. B : percentage of cells that had typical apoptotic morphology. Data are expressed as means ± SD ( n = 6). **Statistically significantly different from the azide group without Zn 2+. The results show that 10 µM Zn 2+ but not 10 µM Co 2+ inhibited tubular cell apoptosis following ATP depletion.

Zn 2+ inhibits caspase activation during ATP depletion. Caspases play a central role in the execution of apoptosis ( 47 ). Thus, to identify the apoptotic events that were blocked by Zn 2+, we initially examined caspase activation. The results are shown in Fig. 2. ATP depletion induced a drastic increase in caspase activity. Zn 2+ at 10 µM attenuated caspase activation in these cells, whereas Co 2+ was less effective. The results were substantiated by in situ detection of caspase activation ( Fig. 2 B ). Here, we conducted immunofluorescence experiments using an antibody that was specific for the active forms of caspase-3 ( 15, 53 ). Clearly, ATP depletion by azide induced the formation of active caspase-3 in many cells, which was significantly suppressed by Zn 2+ but not by Co 2+.

Fig. 2. Inhibition of caspase activation during ATP depletion by Zn 2+. Cells were incubated in glucose-free medium and then returned to full culture medium for 2 h of reperfusion. For the conditions with Zn 2+ or Co 2+, the ions were present during azide incubation and reperfusion. AFC, 7-amino-4-trifluoromethylcoumarin. A : caspase activity measured by enzymatic assays using DEVD.AFC as the substrate. Data are expressed as means ± SD ( n = 4). **Statistically significantly different from the azide group without Zn 2+. B : immunofluorescence (IF) of active casapse-3. Cells were fixed and processed for immunofluorescence using an antibody that specifically reacted with the active fragments of caspase-3. The results show that Zn 2+ suppressed caspase activation in tubular cells following ATP depletion.

Maximal inhibition is shown for Zn 2+ when added during ATP depletion. Previous work by this and other laboratories demonstrated that apoptosis of tubular cells induced by severe ATP depletion is mediated by the intrinsic mitochondrial pathway ( 10, 12, 14 - 16, 27, 32, 40, 51 ). In this experimental model, mitochondrial events of apoptosis and caspase activation occur during ATP depletion. However, apoptotic morphology develops only after the cells are returned to full culture medium for reperfusion. We showed that Zn 2+, when added during ATP depletion and reperfusion, had a potent inhibitory effect on apoptosis ( Figs. 1 and 2 ). To determine whether Zn 2+ affected upstream apoptotic signaling, we tested the effects of Zn 2+, when added at three different time periods: pre-, during, and postazide incubation. The results are summarized in Fig. 3. Maximal apoptosis inhibitory effects of Zn 2+ were shown, when the ion was present during azide incubation or ATP depletion ( Fig. 3 A ). Pretreatment with Zn 2+ for 2 h had a marginal effect, while no effects were shown for Zn 2+ when added after ATP depletion or during reperfusion. Consistently, Zn 2+ added during ATP depletion had maximal inhibitory effects on caspase activation ( Fig. 3 B ). Together, the results suggest that Zn 2+ may inhibit apoptosis by blocking critical steps initiated during ATP depletion.

Fig. 3. Effects of Zn 2+ on apoptosis when added before, during, or after ATP depletion. Cells were incubated with 10 mM azide for 3 h in glucose-free medium and recovered in full culture medium for 2 h. For preazide treatment, the cells were exposed to Zn 2+ for 2 h before azide incubation. For the during-azide group, Zn 2+ was present during azide incubation. For the postazide group, Zn 2+ was present during the reperfusion period. The percentage of cells that developed typical apoptotic morphology ( A ) and caspase activity ( B ) was assessed. Data are expressed as means ± SD ( n = 3). **Statistically significantly different from the azide group without Zn 2+. The results show that maximal inhibitory effects of Zn 2+ were obtained when the ion was present during ATP depletion.

Marginal inhibitory effects of Zn 2+ on Bax activation and cytochrome c release during ATP depletion. We and others showed that tubular cell apoptosis following severe ATP depletion is mediated by the intrinsic mitochondrial pathway ( 10, 12, 14 - 16, 27, 32, 40, 51 ). Specifically, the proapoptotic protein Bax is activated and translocated to mitochondria, where it oligomerizes and presumably forms pathological pores, releasing apoptogenic factors including cytochrome c. Cytochrome c, after being released into the cytosol, associates with Apaf-1, recruiting caspase-9 to form the caspase-activating complex called apoptosome. Thus, to identify the apoptotic events that were blocked by Zn 2+, we first tested whether Zn 2+ inhibited the early apoptotic signaling in mitochondria, i.e., Bax translocation and cytochrome c release. As shown in Fig. 4 A, in control cells, Bax was detected mainly in the cytosolic fraction ( lane 1 ). After 3 h of ATP depletion by azide, large amounts of Bax moved to the membrane-bound fraction enriched with mitochondria ( lane 2 ). Zn 2+ had a marginal effect on Bax translocation; as a result, significant Bax was still detected in the membrane fraction ( lane 3 ). The immunoblot results were confirmed by immunofluorescence analysis. As shown in Fig. 4 B, Bax in control cells had a cytoplasmic staining. ATP depletion induced the association of Bax with mitochondria in many cells, showing a perinuclear punctate staining. Particularly, some of the cells had an intense Bax signal in mitochondria, suggesting membranous insertion and conformational change of the molecule. In the presence of 10 µM Zn 2+, we still detected a large population of cells with intense Bax signal in mitochondria. To quantify Bax activation, we counted the cells that had intense mitochondria staining of Bax. The results show that, following ATP depletion, 45.1% of cells had intense Bax in mitochondria, whereas 33.8% for the group with Zn 2+. We further examined the effects of Zn 2+ on cytochrome c release ( Fig. 5 ). Our immunoblot analyses indicate that ATP depletion induced the release of cytochrome c from mitochondria to cytosol ( lane 2 ), which was suppressed to certain extents but not completely by Zn 2+ ( lane 3 ). The conclusion was supported by immunofluorescence analysis, as shown in Fig. 5 B. Quantitative data show that 53.4% of cells released cytochrome c during ATP depletion. In the presence of Zn 2+, 32.9% of cells showed cytochrome c release.

Fig. 4. Effects of Zn 2+ on Bax translocation during ATP depletion. Cells were incubated with 10 mM azide for 3 h in glucose-free medium. A : immunoblot analysis of Bax. The cells were fractionated into cytosolic and membrane fractions for immunoblot analysis of Bax. B : immunofluorescence of Bax. The cells were fixed and processed for immunofluorescence. C : percentage of cells showing Bax translocation. Cells in the immunofluorescence images were examined to estimate the percentage of cells that had intense Bax staining in mitochondria (Mito). Data are expressed as means ± SD ( n = 3). The results show that Zn 2+ had marginal inhibitory effects on Bax translocation during ATP depletion.

Fig. 5. Effects of Zn 2+ on mitochondrial cytochrome c release during ATP depletion. Cells were incubated with 10 mM azide for 3 h in glucose-free medium. A : immunoblot analysis. The cells were fractionated into cytosolic and membrane fractions for immunoblot analysis of cytochrome c. B : immunofluorescence of cytochrome c. The cells were fixed and processed for immunofluorescence. C : percentage of cells showing cytochrome c release. Cells in the immunofluorescence images were examined to estimate the percentage of cells that had diffuse cytosolic staining. Data are expressed as means ± SD ( n = 3). **Statistically significantly different from the azide group without Zn 2+. The results show that Zn 2+ partially inhibited cytochrome c release during ATP depletion.

Zn 2+ blocks cytochrome c-stimulated caspase activation in isolated cytosol. We showed that Zn 2+ at 10 µM almost completely blocked apoptosis ( Fig. 1 ) but only modestly inhibited Bax translocation and cytochrome c release ( Figs. 4 and 5 ). It is suggested that Zn 2+ may also inhibit apoptosis at levels downstream of cytochrome c release. An immediate step downstream of cytochrome c release is the association of cytochrome c with Apaf-1, recruiting caspase-9 to form the caspase-activation complex, apoptosome. Our previous work examined these events by adding exogenous cytochrome c to isolated cytosol to activate caspases ( 12, 14, 15, 51 ). To test the effects of Zn 2+, we isolated cytosol from control cells. Same amounts of cytochrome c were then added to the cytosol, in the presence or absence of Zn 2+. Caspase activation after adding cytochrome c was measured by enzymatic assays. The results are shown in Fig. 6. Clearly, cytochrome c stimulated caspase activation in isolated cell cytosol. Of significance, impressive inhibitory effects were demonstrated for Zn 2+ in this system. The inhibition by Zn 2+ was evident at 5 µM and complete at 10 µM or higher concentrations ( Fig. 6 ).

Fig. 6. Zn 2+ inhibition of cytochrome c -stimulated caspase activation in isolated cytosol. Cytosol was isolated from normal control cells. Exogenous cytochrome c was then added into the cytosol to induce caspase activation, in the presence or absence of various amounts of Zn 2+. The reconstitution mixtures were finally added to caspase activity assays to determine caspase activity. Data are expressed as means ± SD ( n = 4). The results show that Zn 2+ abolished cytochrome c -stimulated caspase activation in isolated cytosol at low-micromolar levels.

Inhibition of recombinant caspase-3 by Zn 2+. To determine whether Zn 2+ had direct inhibitory effects on caspases, we tested purified recombinant caspase-3. Caspase-3 was incubated with the peptide substrate DEVD.AFC, in the presence or absence of Zn 2+. The cleavage of DEVD.AFC was monitored during the incubation. As shown in Fig. 7, Zn 2+ suppressed the enzymatic activity of caspase-3 in a dose-dependent manner; 50% inhibition was obtained at 10 µM.

Fig. 7. Inhibition of the enzymatic activity of recombinant caspase-3 by Zn 2+. Same amounts of purified recombinant caspase-3 were added to a reaction buffer containing DEVD.AFC in the presence or absence of various amounts of Zn 2+. The cleavage of DEVD.AFC was then monitored to determine the effects of Zn 2+. The values are the average of 3 separate measurements; error bars are omitted for clarity. The results show that Zn 2+ inhibited the enzymatic activity of recombinant caspase-3.

DISCUSSION

This study was designed to provide a systematic analysis of Zn 2+ inhibition of apoptosis in renal tubular cells following ATP depletion. The results show that Zn 2+ suppressed tubular cell apoptosis at low-micromolar concentrations. Zn 2+ inhibited the development of apoptotic morphology as well as the activation of caspases, suggesting that upstream events of apoptosis were attenuated. Consistently, our experiments show that Bax translocation and cytochrome c release during ATP depletion were partially inhibited by Zn 2+. Moreover, in in vitro reconstitution experiments, Zn 2+ was able to suppress cytochrome c -induced caspase activation in isolated cytosol. Finally, Zn 2+ was shown to directly inhibit the enzymatic activity of purified recombinant caspase-3. Together, the results suggest that Zn 2+ is a potent inhibitor of tubular cell apoptosis following ATP depletion. Inhibition of apoptosis by Zn 2+ occurs at multiple levels, on the mitochondria and in the cytosol.

In our experimental model, complete inhibition of apoptosis was achieved by 10 to 50 µM Zn 2+. At the same concentrations, Zn 2+ could only partially inhibit Bax activation and cytochrome c release from the organelles. Quantitative data indicate that, in the presence of 10 µM Zn 2+, only 10% cells developed apoptotic morphology following ATP depletion ( Fig. 1 ). However, over 30% cells in these groups had Bax activation and cytochrome c release. Thus a large population of cells ( 20%) experienced apoptotic alterations at the mitochondrial level, whereas further progression into the apoptotic cascade was blocked by Zn 2+. This observation is at discrepancy with a recent report, which showed that Zn 2+ inhibited the intrinsic pathway of apoptosis at the mitochondria and blocked extrinsic apoptosis at higher concentrations by inhibiting caspases ( 19 ). Obviously, the discrepancy between these two studies might be caused by differences in the experimental models. First, in the earlier study, apoptosis was induced by anisomycin, a potent MAP kinase activator that also inhibits protein translation. Therefore, in addition to direct effects on the apoptotic pathway, Zn 2+ may target early signals triggered by anisomycin, e.g., specific MAP kinases or their substrates. Second, the amounts of Zn 2+ used in the earlier study (0.3-3 mM) were much higher than those used in the current study. At higher concentrations, the specificity of Zn 2+ might be significantly diminished. In our experimental model, 10-50 µM Zn 2+ protected tubular cells, whereas 100 µM Zn 2+ was already toxic, leading to cell detachment, injury, and death in the form of necrosis (data not shown).

Due to technical difficulties, the current study did not measure intracellular concentrations of Zn 2+. Nevertheless, the specific conditions of the experimental model would allow us to speculate that an equilibrium across the plasma membrane might have reached. In these experiments, cells were depleted of ATP for 3 h. In the absence of ATP, the activity of ion channels and pumps was expected to decrease drastically. As a result, passive diffusion and transport of Zn 2+ from the large reservoir of incubation medium would saturate the intracellular Zn 2+ -binding sites, leading to a quick equilibration between the intra- and extracellular spaces. Thus we expect that incubation of ATP-depleted cells with 10-50 µM Zn 2+ would result in similar levels of Zn 2+ within the cells.

It remains unclear as to how micromolar Zn 2+ inhibits Bax activation and cytochrome c leakage during ATP depletion of tubular cells. Dual immunofluorescence of Bax and cytochrome c in the same cells indicated that Bax activation was always accompanied by cytochrome c release (data not shown). This result is consistent with our previous results from related models ( 16, 32, 40, 55 ), supporting a critical role for Bax in cytochrome c release. Thus the question is, how can Zn 2+ prevent Bax translocation and insertion into mitochondria? Apparently, the answer to this question depends largely on our understanding the mechanisms of Bax activation. Well known for its proapoptotic nature, Bax contains three Bcl-2 homology (BH) domains ( 1, 22 ). At its COOH terminus, Bax has a hydrophobic transmembrane domain. In nonapoptotic cells, this domain is covered by the folding NH 2 terminus. Removal of the NH 2 terminus results in the targeting of Bax to mitochondria, suggesting a pivotal role for the exposure of the hydrophobic domain in Bax activation ( 21 ). What controls the folding or unfolding of Bax is a hot spot of debate. In that regard, proteolytic cleavage of Bax usually does not occur in apoptosis; thus removal of the NH 2 terminus cannot be a common mechanism for Bax activation ( 1, 22 ). Currently, there are at least two hypotheses regarding the regulation of Bax; each is supported by significant evidence ( 30 ). In the first hypothesis, Bax is proposed to interact with a regulatory protein. Modifications of the interaction may lead to conformational changes in Bax and the exposure of the hydrophobic domain. A potential Bax-interacting protein is Bid, another proapoptotic Bcl-2 family protein with only one BH domain ( 1, 22 ). We showed that Bid transfection led to Bax translocation, insertion, and oligomerization in mitochondria, followed by cytochrome c release and apoptosis. Cotransfection of Bcl-2 prevented Bid insertion into mitochondrial membranes, and consequently Bax activation was inhibited ( 55 ). Moreover, we showed recently that Bid was activated in renal tubular cells following ATP depletion in vitro and renal ischemia in vivo ( 53 ). The results suggest a possible regulation of Bax and apoptosis by Bid under conditions of tubular cell ischemia. The second hypothesis on the mechanisms of Bax activation emphasizes a role for alterations of the cytosolic environment ( 30 ). In particular, pH changes in the cytosol might be critical. A shift of intracellular pH toward either alkalization or acidification has been linked to conformational changes in Bax, followed by insertion of the molecule into mitochondrial membranes ( 26, 46 ). Apparently, these two hypotheses are not mutually exclusive. For example, the interactions between Bax and its partnering proteins might well depend on the availability of an appropriate cytosolic environment. Whether Zn 2+ inhibits Bax activation through the regulation of specific molecules (e.g., Bid) or the cytosolic environment or both remains to be determined. In addition to these considerations, Zn 2+ is known to affect the physical and chemical properties of cell membranes, for example, the fluidity ( 50 ). It would be interesting to investigate whether these changes make the mitochondrial membrane less suitable for Bax insertion or oligomerization.

Nevertheless, the inhibition of Bax activation and cytochrome c release by low-micromolar Zn 2+ was partial at best, yet the inhibition of apoptosis by the ion was almost complete. The results suggest that apoptotic events downstream of cytochrome c release may also be inhibited by Zn 2+. An immediate step following cytochrome c release is the formation of apoptosome, a protein complex that activates caspases. We tested the effects of Zn 2+ on this apoptotic step, using an in vitro reconstitution system. In this experiment, we isolated cytosol from normal control cells that was free of cytochrome c. When exogenous cytochrome c was added to the cytosol, it bound Apaf-1 to recruit caspase-9 and induced a drastic activation of caspases. Our results show clearly that Zn 2+ was able to block cytochrome c -induced caspase activation in isolated cytosol at low-micromolar levels ( Fig. 6 ). The structure of apoptosome is quite complex, and its regulation is largely unknown. Whether Zn 2+ inhibits the assembly of apoptosome or interferes with its function remains to be investigated. Our results further show that Zn 2+ could directly inhibit the enzymatic activity of caspases ( Fig. 7 ). This observation is supported by previous work ( 43, 45 ). It is interesting that complete inhibition of recombinant caspase-3 required 100 µM Zn 2+, which was 10-fold higher than the amounts of Zn 2+ that were needed to abolish apoptosis in cells and cytochrome c -stimulated caspase activation in isolated cytosol. The results suggest that the primary cause of apoptosis inhibition by Zn 2+ may not be the direct inhibition of caspases; rather, its effects on upstream events including apoptosome can be more critical.

The fact that micromolar Zn 2+ inhibits apoptosis in tubular cells following ATP depletion suggests the potential use of Zn 2+ as a protective agent in vivo during renal ischemia-reperfusion. Indeed, Zn 2+ was protective in ischemic kidneys in rats ( 37 ), although whether it prevented apoptosis was not examined. For in vivo use, one needs to keep in mind that Zn 2+ becomes toxic at higher concentrations. In our experimental model, Zn 2+ induced cell injury and death at concentrations of 100 µM or above (data not shown). Therefore, for in vivo studies, the doses of Zn 2+ need to be carefully controlled and titrated.

In conclusion, this study has documented the potent protective effects of Zn 2+ against tubular cell apoptosis following ATP depletion. Apparently, Zn 2+ targets multiple steps in the apoptotic cascade, involving protection at the mitochondrial level and in the cytosol. These results suggest that Zn 2+ might be considered for renal protection during in vivo ischemia-reperfusion.

GRANTS

Z. Dong is a recipient of Carl W. Gottschalk Research Scholar of the American Society of Nephrology. This work was supported in part by grants from the National Institutes of Health, American Society of Nephrology, and Department of Veterans Affairs.

ACKNOWLEDGMENTS

We thank Dr. A. Srinivasan at Idun Pharmaceuticals (La Jolla, CA) for the antibody to active caspase-3.

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作者单位：Departments of 1 Cellular Biology and Anatomy and 2 Physiology, Medical College of Georgia, Augusta 30912; and 3 Medical Research Service, Department of Veterans Affairs Medical Center, Augusta, Georgia 30904