Bid activation in kidney cells following ATP depletion in vitro and ischemia in - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:14 |只看该作者 |倒序浏览 |打印

作者：Qingqing Wei, Mohammad M. Alam, Mong-Heng Wang, Fushin Yu, and Zheng Dong作者单位：Departments of 1 Cellular Biology and Anatomy and 2 Physiology, Medical College of Georgia, Augusta, Georgia 30912
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      【摘要】
   Bid is a proapoptotic Bcl-2 family protein, which on activation translocates to mitochondria and induces damage to the organelles. Activation of Bid depends on its proteolytic processing into truncated forms of tBid. Bid is highly expressed in the kidneys; however, little is known about its role in renal pathophysiology. In this study, we initially examined Bid activation in cultured rat kidney proximal tubular cells following ATP depletion. The cells were depleted of ATP by azide incubation in the absence of metabolic substrates and then returned to normal culture medium for recovery. Typical apoptosis developed during recovery of ATP-depleted cells. This was accompanied by Bid cleavage, releasing tBid of 15 and 13 kDa. Bid cleavage was abolished in cells overexpressing Bcl-2, an antiapoptotic gene. It was also suppressed by caspase inhibitors. Peptide inhibitors of caspase-9 were more effective in blocking Bid cleavage compared with inhibitors of caspase-8 and caspase-3. Provision of glucose, a glycolytic substrate, during azide incubation inhibited Bid cleavage as well, indicating that Bid cleavage was initiated by ATP depletion. Consistently, Bid cleavage was also induced following ATP depletion by hypoxia or mitochondrial uncoupling. Of significance, cleaved Bid translocated to mitochondria, suggesting a role for Bid in the development of mitochondrial defects in ATP-depleted cells. Finally, Bid cleavage was induced during renal ischemia-reperfusion in the rat. Together, these results provide the first evidence for Bid activation in kidney cells following ATP depletion in vitro and renal ischemia in vivo.
      【关键词】 apoptosis caspase acute renal failure ischemiareperfusion
   ISCHEMIC INJURY TO the kidneys leads to acute renal failure, which is associated with high mortality ( 23 ). Although the development of ischemic acute renal failure involves multiple factors and may proceed in several phases, it is ultimately precipitated by sublethal and lethal damage to the tubular cells ( 13, 22, 38 ). Under these conditions, massive cell death in the necrotic form can be identified, and as a result, termed acute tubular necrosis. On the other hand, recent studies have suggested an apoptotic component in ischemic injury to the kidneys ( 5, 35 ). By morphology, apoptotic cells were identified in ischemia-reperfused kidneys ( 33 ). Biochemically, renal ischemia-reperfusion led to the expression and activation of caspases, a unique family of cysteine proteases responsible for disassembly of apoptotic cells ( 17 ). Endonuclease activation was also documented ( 4 ). In addition, regulation of apoptotic regulatory genes including Bcl-2 and Bax has been shown in ischemia-reperfused kidneys ( 3, 14 ). Finally, several pharmacological agents appeared to ameliorate renal tissue damage during ischemia-reperfusion, at least in part, by diminishing apoptosis ( 9, 18, 19 ). Despite these important observations, the molecular basis underlying apoptosis during renal ischemia-reperfusion remains largely unknown ( 5, 35 ).

Apoptosis is a highly regulated process that plays a central role in the maintenance of tissue homeostasis ( 31 ). Originally defined by cell morphology, apoptosis has now been characterized at the biochemical and molecular level. Two major pathways of apoptosis have been delineated ( 15 ). In the extrinsic pathway, death ligands bind to death receptors, leading 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, followed by the release of apoptogenic molecules such as cytochrome c ( 6 ). In the cytosol, cytochrome c associates with Apaf-1, resulting in the recruitment and activation of caspase-9. These two pathways, triggered differently, converge at the level of executioner caspases, promoting the development of stereotypical features of apoptotic morphology ( 15 ).# J; A/ x, o! _
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Important regulators of apoptosis include Bcl-2 family proteins ( 1, 16 ). Defined by the presence of Bcl-2 homology (BH) domains, these proteins can be proapoptotic or antiapoptotic. Specific function of individual members is determined by the presence and organization of the BH domains. For example, antiapoptotic members, such as Bcl-2 and Bcl-XL, contain four BH domains, whereas some proapoptotic molecules, such as Bax and Bak, contain three (BH1-3) and others only one, the BH3 domain ( 1, 16 ).( B% ~( O- p# b/ D% j* ]# w3 j$ T

Bid is a unique BH3 domain-only proapoptotic protein ( 36 ). Unlike others, Bid activation depends on the proteolytic processing of intact Bid into truncated forms of tBid. tBid, generated thereby, translocates to mitochondria and leads to disruption of the organelles and the release of apoptogenic molecules such as cytochrome c ( 20, 24 ). Bid processing can be conducted by several proteases; however, caspase-8 has been shown to be the major protease responsible for Bid cleavage during death receptor-mediated apoptosis ( 41 ). Caspase-8-mediated Bid processing therefore bridges the extrinsic death receptor-mediated pathway of apoptosis to the intrinsic mitochondrial pathway ( 41 ). This provides a mechanism to amplify the execution signal and exacerbate the pace of cell demise ( 21, 43 ).

Bid is expressed in an abundant manner in the kidneys ( 36 ). However, no information has been available regarding a pathophysiological role of this important proapoptotic molecule in renal cells. On the other hand, recent studies identified a central role for Bid in neuronal cell death during brain ischemia ( 30, 42 ). Under these considerations, we examined Bid activation in an in vitro model of renal cell ATP depletion and in an in vivo model of rat kidney ischemia-reperfusion. In the in vitro model, Bid was cleaved into truncated forms of tBid, following ATP depletion. tBid translocated to mitochondria. Bid activation was attenuated by Bcl-2 overexpression and was also suppressed by caspase inhibitors. In the in vivo model, Bid cleavage into tBid was demonstrated during renal ischemia as well as early reperfusion. Together, these results suggest the potential involvement of Bid in renal cell injury during ischemia-reperfusion.
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MATERIALS AND METHODS
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Materials. Rat kidney proximal tubular epithelial cells were from Dr. U. Hopfer at Case Western Reserve University, Cleveland, OH. The cells were maintained as described ( 39 ). Bcl-2-overexpressing cells were obtained by stable transfection with the Bcl-2 expression vector p actBcl-2 as described previously ( 32 ). The vector was a gift from Dr. J. Yuan at Harvard Medical School, Boston, MA. The rabbit polyclonal antibody against Bid was a gift from Dr. X. M. Yin at the University of Pittsburgh School of Medicine, Pittsburgh, PA. 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). Reagents were also purchased from the following sources: cell-permeable peptide inhibitors of caspases (VAD, IETD, LEHD, DQMD) from Enzyme Systems Products (Dublin, CA); carbonyl cyanide- m -chlorophenyl hydrazone (CCCP) from Calbiochem-Novabiochem (San Diego, CA); all secondary antibodies from Jackson ImmunoResearch (West Grove, PA); and other reagents from Sigma (St. Louis, MO).# G8 r) [! x) k. l
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Renal ischemia. Male Sprague-Dawley rats of 250-275 g were purchased from Charles River. For experiments, the animals were anesthetized with one intraperitoneal injection of 50 mg/kg pentobarbital sodium and placed on a homeothermic table to maintain core body temperature of 37°C. Flank incisions were made to expose both renal pedicles for clamping to induce ischemia. For ischemiaonly group, kidneys were harvested at the end of 35 min of clamping. For reperfusion groups, clamps were released after 35 min of clamping and kidneys were collected after indicated periods of reperfusion. Color changes of the kidneys during the initiation of clamping and release of the clamps were monitored to indicate renal ischemia and reperfusion. Sham operation was conducted by an identical procedure but without clamping of the renal pedicles. Serum creatinine levels were determined to indicate renal function, using a kit purchased from Stanbio Laboratory (Boerne, TX).
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ATP depletion. Three approaches were used to deplete the cells of ATP. First and in the majority of the experiments, cells were subjected to 10 mM azide incubation 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 ). In the second approach, cells were incubated in glucose-free Krebs-Ringer bicarbonate solution with 15 µM CCCP, a mitochondrial uncoupler ( 10 ). In the third approach, cells were subjected to severe hypoxia in glucose-free Krebs-Ringer bicarbonate solution ( 12, 32 ). To remove residual oxygen and maximize the degree of hypoxia, EC Oxyrase, a biocatalytic oxygen-reducing agent, was added at 1.2 U/ml in the incubation medium. After ATP depletion for indicated periods, groups of cells were returned to the full culture medium to simulate in vivo reperfusion.
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Morphological examination of apoptosis. Cells were stained for 2-5 min with 5 µg/ml of Hoechst 33342 in PBS at room temperature. Cellular morphology was monitored by phase-contrast microscopy. Nuclear staining by Hoechst 33342 was examined by fluorescence microscopy. Typical apoptotic morphology included cellular shrinkage, nuclear condensation and fragmentation, and formation of apoptotic bodies.
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Immunofluorescence of active caspase-3. Indirect immunofluorescence was performed as described previously with modifications ( 11, 32 ). Briefly, cells were grown on collagen-coated glass coverslips for experiments. After incubation, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were incubated for 1 h in a blocking buffer containing 2% normal goat serum. The cells were subsequently incubated for 1 h with the primary antibody, a rabbit polyclonal antibody specific for active caspase-3 ( 34 ). Finally, the cells were exposed to a Cy3-labeled goat anti-rabbit secondary antibody. Staining was examined by fluorescence microscopy.

Immunoblot analysis. Proteins were analyzed by immunoblotting using NuPAGE Gel Systems as described previously ( 11, 12, 32 ). Briefly, samples were resolved under reducing conditions on 10 or 12% Bis-Tris gels in MES running buffer and electroblotted onto polyvinylidene difluoride membranes. The blots were blocked with 5% fat-free milk and then exposed to the primary antibody overnight at 4°C. Finally, the blots were incubated with the horseradish peroxidase-conjugated secondary antibody, and antigens on the blots were revealed by reactions with chemiluminescent substrates (Pierce, Rockford, IL). The reactivity of the antibody was significantly lower for tBid than that for intact Bid. Thus the same blots were exposed separately to reveal Bid and tBid, with longer exposure for tBid.
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Cellular fractionation. To analyze the intracellular distributions of Bid and tBid, cells were fractionated into cytosolic and membrane-bound organellar fractions enriched with mitochondria using low concentrations of digitonin. Digitonin at low concentrations selectively permeabilizes the plasma membranes, without solubilizing mitochondria. This method has been successfully used by us and others to study protein translocations during apoptosis ( 26, 29, 32, 37 ). Briefly, cells were exposed to 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 to collect the soluble part as the cytosolic fraction. The digitonin-insoluble part was further extracted with 2% SDS buffer to collect the membrane-bound organellar fraction. Because Bid and tBid redistribution mainly takes place between the cytosol and mitochondria ( 20, 24 ), immunoblot analysis of the organellar fraction is expected to reveal mitochondrial content of the molecules.: A% h9 K2 E+ z
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Apoptosis during recovery of ATP-depleted cells. An important determinant of ischemic cell injury is the depletion of cellular ATP ( 23 ). Thus to model renal ischemia-reperfusion, we subjected cultured rat kidney proximal tubular cells to azide treatment in the absence of metabolic substrates to induce ATP depletion. ATP-depleted cells were subsequently returned to full culture medium for recovery or "reperfusion." Significant amounts of apoptosis developed within a few hours of recovery. As shown in Fig. 1 A, many cells exhibited typical apoptotic morphology with a shrunk configuration and apoptotic bodies. Nuclear condensation and fragmentation were also evident ( Fig. 1 B ). Of interest, caspase activation was demonstrated in the same population of cells. As shown by immunofluorescence, the active form of caspase-3 was revealed specifically in cells with typical apoptotic morphology ( Fig. 1 C ). Vital dye exclusion assays suggested that necrotic cell death under the experimental conditions was very limited (not shown). In general, 40% cells underwent apoptosis during 2 h of recovery period following 3 h of azide treatment. Without azide exposure, control cells had less than 5% apoptosis ( Fig. 1 D ). These experiments established a relatively "clean" in vitro model to study apoptotic mechanisms of ischemic renal injury.

Fig. 1. Apoptosis during recovery of ATP-depleted cells. Cultured rat kidney proximal tubular cells were incubated with 10 mM azide for 3 h in a glucose-free physiological buffer and then returned to full culture medium for 2 h of recovery. The cells were fixed for immunofluorescence analysis of active caspase-3 and nuclear staining with Hoechst 33342, as described in MATERIALS AND METHODS. Images of the same fields of cells were obtained by phase-contrast microscopy and fluorescence microscopy. Arrows highlight the cells that exhibited typical apoptotic morphology and immunofluorescence staining of active caspase-3. Also included is a phase-contrast image of control cells without azide treatment ( D ). The results show that typical apoptotic morphology developed during recovery or reperfusion of ATP-depleted cells. Importantly, the same cells displayed caspase activation./ D/ X: `' v2 w! S5 T

Bid cleavage during apoptosis triggered by azide. To determine whether Bid was activated during apoptosis of renal cells, we first analyzed Bid cleavage by immunoblotting, because Bid activation depends on its proteolytic processing into truncated forms of tBid. For this purpose, cells were incubated with azide in glucose-free Krebs buffer for 0-3 h alone, or followed by recovery in full culture medium. Whole cell lysates were collected in Lameali buffer containing 2% SDS and analyzed by immunoblotting using a polyclonal antibody against Bid. The results are shown in Fig. 2. In control cells, intact Bid with an apparent size of 22 kDa was detected ( lane 1 ). Bid was not processed during azide treatment ( lanes 2 - 4 ). In sharp contrast, upon reperfusion or recovery, Bid was cleaved into truncated forms of tBid, which showed apparent sizes of 15 and 13 kDa ( lanes 5 - 7 ). As a result, intact Bid decreased in the reperfused cells, accompanied by the appearance of 15 and 13 kDa tBid. Of note, reactivity of the antibody was significantly lower for tBid than that for intact Bid. Thus the same blots were exposed separately to reveal Bid and tBid, with longer exposure for tBid. Nevertheless, decreases in intact Bid and concomitant production of tBid indicated Bid processing during recovery of ATP-depleted cells.  R. `$ j/ U* G3 S5 R

Fig. 2. Bid processing during recovery of ATP-depleted cells. Renal proximal tubule cells (RPTC) were incubated with 10 mM azide in a glucose-free physiological buffer for 0 to 3 h alone or followed by recovery in full culture medium for 0 to 3 h. Whole cell lysates were extracted with Laemmli buffer containing 2% SDS and subjected to immunoblot analysis of Bid using a polyclonal antibody. Bid was shown to be cleaved into 15- and 13-kDa fragments of tBid during recovery, after azide treatment ( lanes 5 - 7 ). In sharp contrast, no significant Bid cleavage was detected in control cells ( lane 1 ) or during azide incubation alone ( lanes 2 - 4 ). Of note, as indicated in MATERIALS AND METHODS, the antibody reacted at significantly higher affinity with full-length Bid of 22 kDa, compared with the truncated Bid of 15 and 13 kDa. Thus the blots were usually subjected to low exposure to reveal the 22-kDa Bid and high exposure to reveal the 15- and 13-kDa tBid, respectively. Similar two-step exposure was employed for experiments described in subsequent figures.- W6 e# h2 Q" u* r

Bid cleavage following hypoxia or CCCP treatment. To substantiate our observation that Bid was proteolytically processed during recovery of ATP-depleted cells, we tested other models of ATP depletion, namely, hypoxia and CCCP treatment. Severe hypoxia or anoxia leads to the arrest of cellular respiration and ATP production by oxidative phosphorylation. CCCP, on the other hand, dissipates mitochondrial membrane potential and as a result uncouples respiration from ATP generation in the organelles. As expected, in the absence of glycolytic substrates, both severe hypoxia and CCCP led to rapid declines of cellular ATP (data not shown). As for the azide-treated group, after 5 h of hypoxia or 2 h of CCCP incubation, cells were returned to the full culture medium for recovery. The results are shown in Fig. 3. Clearly, Bid cleavage was detected during recovery of the ATP-depleted cells, irrespective of the approaches for ATP depletion. Again, intact Bid decreased, which was accompanied by the production of tBid ( lanes 2 - 4 ).
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Fig. 3. Bid processing during reoxygenation of hypoxic cells and recovery of carbonyl cyanide-m-chlorophenyl hydrazone (CCCP)-treated cells. RPTC (control: lane 1 ) were subjected to 3 h of azide treatment, 5 h of hypoxia, or 2 h of CCCP (a mitochondrial uncoupler) incubation in glucose-free buffers, followed by 2 h of recovery in full culture medium. Whole cell lysates were collected for immunoblot analysis of Bid. Clearly all 3 treatments of ATP depletion induced Bid cleavage, releasing tBid of 15 and 13 kDa ( lanes 2 - 4 ).( _: O/ j4 [, h$ C1 a
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Inhibition of Bid cleavage by caspase inhibitors and glucose. To gain insights into the mechanisms underlying Bid cleavage following ATP depletion, we initially tested the effects of VAD, a broad-spectrum peptide inhibitor of caspases ( 11 ). The same experiment also tested the effects of glucose, the substrate of glycolysis, and N -acetylcysteine (NAC), an antioxidant. As shown in Fig. 4 A, inclusion of 50 µM VAD or 5.5 mM glucose during azide incubation suppressed Bid cleavage, whereas 10 mM NAC was not effective. Because glucose facilitated glycolysis and prevented ATP depletion during azide treatment (data not shown), the results provide further support for a role of ATP depletion in triggering Bid cleavage. The results also suggest that Bid cleavage might be mediated by caspases and can be dissociated from oxidative stress or the production of free radicals. To further determine the role of specific caspases in Bid cleavage, we examined the effects of peptide inhibitors with specificity toward caspase-8, caspase-9, or caspase-3 ( Fig. 4 B ). All three inhibitors showed inhibitory effects on Bid cleavage (compare lanes 3 - 11 with lane 1 ). However, the caspase-9 inhibitor LEHD appeared to be most effective. At 20-50 µM, LEHD almost completely blocked Bid cleavage ( lanes 6 and 7 ), whereas Bid cleavage was still evident in the presence of 50 µM IETD and DQMD ( lanes 3 and 9 ), inhibitors of caspase-8 and -3, respectively. LEHD was also more effective in suppressing apoptosis. In the presence of 50 µM LEHD, apoptosis was reduced to 10 20% from 40%. The results suggest an important role for a caspase-9-initiated proteolytic cascade in Bid activation and apoptosis in this experimental model.. W* z& l6 T1 o* {7 _- }

Fig. 4. Inhibition of Bid processing by caspase inhibitors and glucose during azide treatment. A : RPTC (control: lane 1 ) were subjected to 3 h of azide treatment, followed by 2 h of recovery in full culture medium. When indicated, the general caspase inhibitor VAD of 50 µM, the glycolytic substrate glucose of 5.5 mM, or the antioxidant N -acetylcysteine (NAC) of 10 mM was included during azide incubation and recovery. Whole cell lysates were collected for immunoblot analysis of Bid/tBid. The results show that Bid processing was inhibited by VAD and glucose but not by NAC. It is suggested that Bid cleavage was mediated by caspases, of which activation depended on ATP depletion and not oxidative stress. B : RPTC (control: lane 2 ) were subjected to 3 h of azide treatment, followed by 2 h of recovery in full culture medium. When indicated, caspase inhibitors (IETD, LEHD, or DQMD) were present at 10, 20, or 50 µM. Whole cell lysates were collected for immunoblot analysis of Bid/tBid. The results show that these peptide caspase inhibitors suppressed Bid processing, with LEHD being most effective." A* O% {$ Y! g. t9 ]5 x
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Inhibition of Bid cleavage by Bcl-2 expression. To further examine apoptosis-associated Bid cleavage, we compared wild-type proximal tubular cells with the cells stably transfected with Bcl-2, an anti-apoptotic gene. These two types of cells were subjected to 3 h of azide treatment in glucose-free buffer alone or followed by 2 h of recovery in full culture medium. Consistent with previous observations, in wild-type cells, apoptosis developed within a couple of hours of recovery. The development of apoptosis was completely blocked in Bcl-2-transfected cells (data not shown). Of significance, Bid cleavage was also prevented by Bcl-2 overexpression ( Fig. 5 : lanes 3 and 7 ), suggesting a close relationship between the development of apoptosis and Bid processing.

Fig. 5. Inhibition of Bid processing by Bcl-2 overexpression. Wild-type cells and cells overexpressing Bcl-2 were subjected to 3 h of azide treatment followed by 2 h of recovery. Whole cell lysates were extracted for immunoblot analysis of Bid/tBid. In wild-type cells, Bid was proteolytically processed into tBid during recovery following azide incubation ( lane 3 ); Bid processing did not take place in Bcl-2-transfected cells either during or following azide treatment ( lanes 6 and 7 ). The results further suggest that Bid processing might be a result of caspase activation during the development of apoptosis.( d+ V( V: o: S
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Translocation of tBid to mitochondria. The action site for Bid within the cells is the mitochondria. tBid, generated via Bid processing, translocates to mitochondria to induce pathological alterations in the organelles, releasing apoptogenic factors including cytochrome c ( 20, 24 ). To determine whether the cleaved Bid in our experimental model was indeed active, we analyzed the distribution within the cells. To this end, cells were fractionated into cytosolic and membrane-bound organellar fractions enriched with mitochondria. The fractions were subjected to immunoblot analysis of Bid/tBid. The results are shown in Fig. 6. Consistent with previous observations, only intact Bid was detected in control cells, with the majority of Bid shown in the cytosol ( lanes 1 and 3 ). In reperfused cells following azide treatment, intact Bid as well as tBid of 15 and 13 kDa was detected ( lanes 2 and 4 ). Of significance, while the majority of intact Bid existed in the cytosol ( lane 2 ), tBid moved to the mitochondrial fraction ( lane 4 ). The translocation was partial for the 15-kDa tBid and virtually complete for the 13-kDa tBid. The results suggest that Bid after cleavage into tBid became active and may be involved in subsequent mitochondrial disruption.

Fig. 6. tBid translocation to mitochondria. Control RPTC cells (C) and the cells after 2 h of recovery following azide incubation (A) were fractionated into cytosolic (Cyto) and mitochondrial (Mito) fractions for immunoblot analysis of Bid/tBid. In control cells, Bid was intact and was detected mainly in the cytosol ( lanes 1 and 3 ). After 2 h of recovery following azide treatment, Bid was cleaved into tBid of 15 and 13 kDa. tBid of 15 kDa was detected in the cytosol as well as in the mitochondira, whereas the 13-kDa fragment was shown exclusively in the mitochondrial fraction ( lanes 2 and 4 ). The results suggest that Bid after processing into tBid becomes active and may be involved in mitochondrial disruption./ a2 S1 w; y) z& ?$ D1 Q

Bid cleavage during renal ischemia-reperfusion. To extend the in vitro observations to in vivo systems, we analyzed Bid cleavage in a rat model of renal ischemia-reperfusion. The results are shown in Fig. 7. In sham-operated controls, intact Bid of 22 kDa was detected ( lane 1 ). Ischemia of 35 min induced Bid cleavage, releasing tBid of 15 kDa but not the 13-kDa form. Reperfusion of the kidneys for 8 h led to further Bid cleavage. As a result, intact Bid was reduced significantly, accompanied by the production of both 15- and 13-kDa forms of tBid ( lane 3 ). Intact Bid remained low after 24 h of reperfusion, whereas tBid gradually disappeared at this stage ( lane 4 ). After 48 h or longer reperfusion, the levels of intact Bid were restored, without evident cleavage. The results suggest a dynamic Bid regulation during renal ischemia-reperfusion.7 m) H( Y1 x' f  q# T4 O2 q

Fig. 7. Bid processing during renal ischemia-reperfusion. Bilateral renal ischemia of 35 min was induced in Sprague-Dawley rats by renal pedicle clamping, as described in MATERIALS AND METHODS. After ischemia, 1 group of animals was killed to harvest the kidneys ( lane 2 ). For other groups, clamps were released for 8 to 72 h of reperfusion to collect kidneys ( lanes 3 - 6 ). Control animals were subjected to sham operation without renal pedicle clamping ( lane 1 ). Renal tissues were analyzed for Bid/tBid by immunoblotting. The results indicate that Bid was processed into 15-kDa tBid during renal ischemia and was further processed into 13-kDa tBid during early reperfusion ( lanes 2 and 3 ).9 ?5 z% H' z4 r0 c+ n# W8 G
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This study examined Bid activation in an in vitro model of ATP depletion and in an in vivo model of renal ischemia-reperfusion. In the in vitro model, Bid was proteolytically processed into tBid during recovery or reperfusion of ATP-depleted cells. This was accompanied by the development of apoptosis. Expression of Bcl-2, an antiapoptotic gene, inhibited apoptosis as well as Bid processing. Bid processing was also suppressed by caspase inhibitors. Importantly, tBid released during Bid processing translocated to mitochondria, suggesting a role for Bid in the development of mitochondrial pathology. The in vitro results were supported by the in vivo observation that Bid was processed into active forms during renal ischemia and early reperfusion. Together, it is suggested that Bid might be involved in renal cell injury during ischemia-reperfusion." Y: g9 }# v: v( s5 T
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Bid is a proapoptotic member of the Bcl-2 family proteins, which has only one BH domain (BH3) ( 41 ). Different from other BH3-only members, Bid exhibits several unique functional and structural features. Prominent among them is that Bid activation depends on proteolytic processing into active forms of tBid. Several types of proteases were shown to be able to process Bid. In death receptor-mediated apoptosis, caspase-8 is the major protease responsible for Bid cleavage ( 20, 24 ). During myocardial ischemia-reperfusion, Bid processing was shown to be mediated by calpain ( 7 ). Our previous study demonstrated the activation of several types of caspases during ATP depletion induced by severe hypoxia ( 11 ). Consistently, the current study showed that azide-induced apoptosis was suppressed by the general caspase inhibitor VAD and specific peptide inhibitors of caspase-9, -8, and -3. Our subsequent analyses showed that the caspse-9 inhibitor was most effective in blocking Bid processing ( Fig. 4 B ). These results suggest an important role for a caspase-9-initiated proteolytic cascade in Bid processing and the development of apoptosis in this experimental model. Whether caspase-9 per se or downstream caspase proteolyses Bid remains to be determined.& @- f5 x: E9 M9 p: J8 `. v

It is interesting that, although Bid activation in vitro took place only during recovery or reperfusion of ATP-depleted cells ( Fig. 2 ), in vivo Bid processing started from ischemia and extended to early reperfusion ( Fig. 7 ). This observation suggests that the mechanisms responsible for Bid activation in vivo could be far more complex and may involve different proteases at different stages. Such a scenario is consistent with the fact that cell injury during renal ischemia-reperfusion is a multifactorial process, which may proceed as distinguished episodes ( 27 ). During ischemia, increases in intracellular Ca 2  may activate calpain. Meanwhile, mitochondrial damage may also have been initiated, releasing cytochrome c and leading to caspase activation. On activation, calpain and caspases may activate Bid separately or in a collaborative manner. On the other hand, after reperfusion, the death receptor-mediated extrinsic pathway of apoptosis might be important. The activation of the Fas system has been documented during renal ischemia-reperfusion in a model of unilateral occlusion ( 28 ). Presumably, Fas activation leads to the activation of caspase-8, which subsequently protealyses Bid into the active forms of tBid. Of note, Bid activation as indicated by the production of tBid was maximal at early reperfusion and decreased significantly after 24 h of reperfusion ( Fig. 7 ). It would be interesting to study the activation dynamics of calpain, caspase, Fas, and Bid systematically in the same animal model.

An important question is: what does Bid do during renal ischemia-reperfusion? Apparently, without experiments in Bid-deficient cells or Bid-null mice, we can only speculate at this moment. Based on our in vitro results showing tBid translocation to mitochondria, it is suggested that Bid/tBid might promote cell death at the mitochondrial level. Damage to mitochondria, outer membranes in particular, leads to the release of apopotogenic factors including cytochrome c, Smac, Omi, AIF, and endonuclease G ( 25 ). Cytochrome c, after being released into the cytosol, associates with Apaf-1, forming apoptosome to activate caspases ( 6 ). Smac and Omi can sequester IAP, a family of caspase inhibitory proteins, to liberate the caspases for full activation ( 21 ). On the other hand, AIF and endonuclease G released from mitochondria might promote cell death in a caspase-independent manner by facilitating nuclear fragmentation ( 15 ). Although it is unclear how Bid/tBid induces pathological alterations in the mitochondria, recent studies suggested an activation cascade for Bcl-2 family proteins during apoptosis ( 8 ). In this model, BH3 domain-only proteins such as Bid move to mitochondria and activate multi-BH domain proapoptotic proteins such as Bax and Bak, leading to their oligomerization and formation of pathological pores. Antiapoptotic proteins including Bcl-2 and Bcl-XL are positioned to block apoptosis at two separate steps, through the inhibition of Bax/Bak and BH3-only proteins including Bid. Consistent with this scenario, our previous study demonstrated Bax oligomerization in the outer membrane of mitochondria during ATP depletion of rat proximal tubular cells ( 26 ). More recently, we showed that transfection of Bid into HeLa cells led to the activation of Bax and Bak, permeabilizing mitochondria to release cytochrome c. Cotransfection of Bcl-2 prevented the insertion of tBid into mitochondrial membranes and as a result, it suppressed Bax/Bak activation, cytochrome c release, and cell death ( 40 ). Further investigations using Bid-deficient cells or animals should provide insights into the role of Bid in renal cell injury initiated by ATP depletion in vitro and ischemia in vivo.
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In conclusion, this study provided the first evidence for Bid activation in kidney cells following ATP depletion in vitro and renal ischemia-reperfusion in vivo. Considering the important roles played by Bid in both extrinsic and intrinsic pathways of apoptosis, the activation of this molecule suggests its involvement in renal cell injury by ischemia-reperfusion. Genetic and pharmacological approaches targeting Bid or tBid may therefore provide new strategies to ameliorate tissue damage during ischemic acute renal failure.
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ACKNOWLEDGMENTS
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We thank Dr. M. A. Venkatachalam from the University of Texas Health Science Center at San Antonio for advice on the animal model of renal ischemia. We also thank J. Wang for technical assistance in some of the experiments.
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This work was supported in part by a Carl W. Gottschalk Research Scholar grant from the American Society of Nephrology and National Institutes of Health Grant DK-58831.) o5 P4 n) `+ [0 V$ j% T/ G
      【参考文献】
Adams JM and Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 281: 1322-1326, 1998.
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Ashkenazi A and Dixit VM. Death receptors: signaling and modulation. Science 281: 1305-1308, 1998.6 ?5 @% f! d6 O# z, l2 N
+ p8 y0 s3 ?* b  s
- s; d6 p' l7 @0 Z6 k9 l
! m6 T, F; z+ N3 {+ j0 V" g6 F
Basile DP, Liapis H, and Hammerman MR. Expression of bcl-2 and bax in regenerating rat renal tubules following ischemic injury. Am J Physiol Renal Physiol 272: F640-F647, 1997.
  b2 a4 D6 o, D

& @/ q8 h0 l  M' K
Basnakian AG, Ueda N, Kaushal GP, Mikhailova MV, and Shah SV. DNase I-like endonuclease in rat kidney cortex that is activated during ischemia/reperfusion injury. J Am Soc Nephrol 13: 1000-1007, 2002.# w0 H& r: X4 O: Q& e" F) c
: Z: X5 M2 H  l) `9 _4 O  V2 h

( @$ f0 L  E# T% i, z5 v9 L( L$ x4 s
Bonegio R and Lieberthal W. Role of apoptosis in the pathogenesis of acute renal failure. Curr Opin Nephrol Hypertens 11: 301-308, 2002.) F/ A. p7 p9 J2 x4 u
7 B6 l! k8 b4 V7 H3 K. M  u

Budihardjo I, Oliver H, Lutter M, Luo X, and Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15: 269-290, 1999.
- z. Z, y" e: a9 z" C

Chen M, He H, Zhan S, Krajewski S, Reed JC, and Gottlieb RA. Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276: 30724-30728, 2001.

2 \7 I* U$ b4 X: o' S+ s
Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, and Korsmeyer SJ. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8: 705-711, 2001.
: m2 X( D. `- g. Z9 a1 j" V. \& D* ]
0 a- \- X3 F; d  v. h# S$ \8 k/ l
; E) {3 a! D, W/ a: t: f
Daemen MA, van't Veer C, Denecker G, Heemskerk VH, Wolfs TG, Clauss M, Vandenabeele P, and Buurman WA. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104: 541-549, 1999.0 C- X( @7 L( ?' l$ {) a9 h- Z5 L
  X* ~7 T/ D* [, L
7 G5 ], T+ f1 ?) Q
' m- t1 V, C4 j' d  o; w3 O
Dong Z, Patel Y, Saikumar P, Weinberg JM, and Venkatachalam MA. Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine. Lab Invest 78: 657-668, 1998." @4 H2 M! v, Y$ o0 e7 ?, c5 Z

Dong Z, Saikumar P, Patel Y, Weinberg JM, and Venkatachalam MA. Serine protease inhibitors suppress cytochrome c -mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem J 347: 669-677, 2000.
0 w$ ?1 P9 ^2 D1 _4 s( M

' E) l' `5 R& X, L: p
Dong Z, Venkatachalam MA, Wang J, Patel Y, Saikumar P, Semenza GL, Force T, and Nishiyama J. Upregulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms. J Biol Chem 276: 18702-18709, 2001.& ~1 V  Z$ e, b5 d

# ^* j+ I3 _* X+ d0 `* e2 R
Edelstein CL, Ling H, and Schrier RW. The nature of renal cell injury. Kidney Int 51: 1341-1351, 1997.( s" @7 z7 V) b+ o  \8 x" D

8 |& P' B' F- E7 b4 f

Gobe G, Zhang XJ, Willgoss DA, Schoch E, Hogg NA, and Endre ZH. Relationship between expression of Bcl-2 genes and growth factors in ischemic acute renal failure in the rat. J Am Soc Nephrol 11: 454-467, 2000.

& ]( E( b: m9 ?1 Q8 `( z: a

Green DR. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102: 1-4, 2000.
  N7 D9 Y& P3 ]& i# J
* E( O3 s7 h% \# J
5 ]( X9 g  [7 t( j
Gross A, McDonnell JM, and Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13: 1899-1911, 1999.
7 }' _& v" Y% P6 d- O
0 x  k2 u5 t3 s3 ?5 Y- u) E/ k. v
7 T6 K1 ?$ C9 x) R
Kaushal GP, Singh AB, and Shah SV. Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol Renal Physiol 274: F587-F595, 1998.. ?5 U% C8 E; r5 D& x

5 s4 D7 X5 E; }
Kelly KJ, Plotkin Z, and Dagher PC. Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury. J Clin Invest 108: 1291-1298, 2001.
5 D. m9 C) ^9 [$ p. G: [# g# O
. h# q, Z4 @, g- c9 ?8 F0 f3 N4 ~' B

Kelly KJ, Plotkin Z, Vulgamott SL, and Dagher PC. p53 Mediates the apoptotic response to GTP depletion after renal ischemia-reperfusion: protective role of a p53 inhibitor. J Am Soc Nephrol 14: 128-138, 2003.& b+ K% V' [8 T' `) f8 O/ m
; R+ ~& e% I  z( W  ^
8 C) s5 w5 z4 s

Li H, Zhu H, Xu CJ, and Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491-501, 1998.5 R1 T  A( X+ `0 d
9 A3 t( {, W$ _! K3 z, f
8 k3 o8 E& i1 i% G1 _: y, A

Li S, Zhao Y, He X, Kim TH, Kuharsky DK, Rabinowich H, Chen J, Du C, and Yin XM. Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. J Biol Chem 277: 26912-26920, 2002.

1 [2 R% V: V( X! v% x
0 s* c4 x' \+ _
Lieberthal W, Nigam SK, Bonventre JV, Brezis M, Siegel N, Rosen S, Portilla D, and Venkatachalam MA. Acute renal failure. I. Relative importance of proximal vs. distal tubular injury. Am J Physiol Renal Physiol 275: F623-F631, 1998.' ]! I* p4 H- g9 a" p

Lieberthal W, Nigam SK, Molitoris BA, Weinberg JM, Venkatachalam MA, Zager RA, Nath KA, and Goligorsky MS. Acute renal failure. II. Experimental models of acute renal failure: imperfect but indispensable. Am J Physiol Renal Physiol 278: F1-F12, 2000.

6 P6 D( p' g3 S& a: u! J  m7 R
, W7 O% A& y0 b! V
Luo X, Budihardjo I, Zou H, Slaughter C, and Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490, 1998.
3 \: o, [& g2 l' l% z, ^

2 T7 {3 N  N# e
Martinou JC and Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2: 63-67, 2001.1 V' X% ]4 V. G/ o, T
* m1 c8 q: I2 i4 x5 p0 V5 F" [* r: A
1 f' f/ j4 E) {% Y. ]
% T4 ?! P, V, _1 V7 t9 ]
Mikhailov V, Mikhailova M, Pulkrabek DJ, Dong Z, Venkatachalam MA, and Saikumar P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem 276: 18361-18374, 2001." T1 q8 o0 B6 v( }

, B9 |. e, h0 p6 J- r2 D8 m! {4 d+ U/ Z

Molitoris BA. Transitioning to therapy in ischemic acute renal failure. J Am Soc Nephrol 14: 265-267, 2003.# $ C9 r3 D$ ^, X' z- P

5 q7 Z9 z5 l) q6 x

Nogae S, Miyazaki M, Kobayashi N, Saito T, Abe K, Saito H, Nakane PK, Nakanishi Y, and Koji T. Induction of apoptosis in ischemia-reperfusion model of mouse kidney: possible involvement of Fas. J Am Soc Nephrol 9: 620-631, 1998.
1 }8 f3 O$ \: B  `. T: i
: s4 j. J7 r3 P* |3 P) {$ O, V4 P

Nomura M, Shimizu S, Sugiyama T, Narita M, Ito T, Matsuda H, and Tsujimoto Y. 14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem 278: 2058-2065, 2003.# Y& y( m3 c$ n: ]8 p1 M

: U, `' S. Q8 J( $ B& c" f
9 n4 T& ]* A. U# U. m* X* Z! E
Plesnila N, Zinkel S, Le DA, Amin-Hanjani S, Wu Y, Qiu J, Chiarugi A, Thomas SS, Kohane DS, Korsmeyer SJ, and Moskowitz MA. BID mediates neuronal cell death after oxygen/glucose deprivation and focal cerebral ischemia. Proc Natl Acad Sci USA 98: 15318-15323, 2001.; b9 n6 }9 K, n" C

' }( i# G/ x; H1 s2 ~1 }
Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, and Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med 107: 489-506, 1999.
( [; E4 f& i% ]4 Y5 `) J: c$ E/ O  b
, D. W/ Q/ x1 E: N; x
8 g% ^) q+ P$ l2 L( d6 l% G" C
Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, and Venkatachalam MA. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17: 3401-3415, 1998.- G, l- |' Z1 A+ X2 z

3 P, l4 b& l0 v

Schumer M, Colombel MC, Sawczuk IS, Gobe G, Connor J, O'Toole KM, Olsson CA, Wise GJ, and Buttyan R. Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 140: 831-838, 1992.
2 `) c' n6 f7 i0 x6 N

% [6 X* C( ~7 l6 P; ]* ^  H' N* N& U5 ^
Srinivasan A, Roth KA, Sayers RO, Shindler KS, Wong AM, Fritz LC, and Tomaselli KJ. In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ 5: 1004-1016, 1998.% E4 T9 d" L& `3 q, h
. M: j' @0 ~3 f1 S

Ueda N, Kaushal GP, and Shah SV. Apoptotic mechanisms in acute renal failure. Am J Med 108: 403-415, 2000.
3 q4 h9 y3 {/ f

Wang K, Yin XM, Chao DT, Milliman CL, and Korsmeyer SJ. BID: a novel BH3 domain-only death agonist. Genes Dev 10: 2859-2869, 1996.; N& N0 `% i7 e  n% H. f

8 c- c, _9 @5 T, P7 Z" e" |2 B, m
" F8 F! }- i8 ^9 G) |
Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, and Green DR. Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J Cell Biol 153: 319-328, 2001., O- h! A  N$ p6 T
' i2 C! b6 Q* f5 s$ ~  O

Weinberg JM. The cell biology of ischemic renal injury. Kidney Int 39: 476-500, 1991.
# _2 i+ \( v. y8 f- o
; [0 m" b; y# I" ~/ @
5 ]4 g% k( y. g( v8 i9 x
Woost PG, Orosz DE, Jin W, Frisa PS, Jacobberger JW, Douglas JG, and Hopfer U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int 50: 125-134, 1996.

" \$ z8 _% U0 N6 h; k- T

Yi X, Yin XM, and Dong Z. Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed. J Biol Chem 278: 16992-16999, 2003.! J0 B' }- }8 q+ A, U$ a2 ~' @: ]
# L* ?, b( B3 h

Yin XM. Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Res 10: 161-167, 2000.2 |& ]  l  k& m" }/ ]" F

Yin XM, Luo Y, Cao G, Bai L, Pei W, Kuharsky DK, and Chen J. Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia. J Biol Chem 277: 42074-42081, 2002.# o. {: h) `6 a3 b/ J2 [/ I3 l! Q
6 W% w, l( W2 S. i4 Y- v9 }9 t
" z4 t8 A8 Q; X+ R" b- R
& O6 l# n: ]9 |& L7 f7 _9 ^
Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA, and Korsmeyer SJ. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886-891, 1999.

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