INTRODUCTION

TOP ABSTRACT INTRODUCTION MODES OF CELL DEATH MORPHOLOGICAL, BIOCHEMICAL, AND... MORPHOLOGY OF APOPTOSIS MORPHOLOGY OF NECROSIS ENDONUCLEASES AND DNA... MOLECULAR MECHANISMS OF... APOPTOSIS-BASED THERAPEUTIC... MOLECULAR MECHANISMS OF... CELL DEATH IN ISCHEMIC... MECHANISMS OF NECROSIS IN... MOLECULAR MECHANISMS OF... DEPLETION OF GTP DETECTION OF APOPTOSIS IN... CONCLUDING REMARKS REFERENCES

CELLULAR DEATH AND RESORPTION are critical biological processes that are crucial not only to normal histogenesis and organelle turnover but also to the pathogenesis of tissue injury and diseases (67, 73, 99, 141, 188, 199). Normal physiological cell death or programmed cell death (PCD) occurs continuously in proliferating tissues and counterbalances excessive cell proliferation during mitosis. PCD is an integral part of maintaining the normal functioning of the immune system and in sculpting the early embryo (67, 188, 199). Nonsynchronized apoptosis can result in pathophysiological outcome and is implicated in causing a variety of human diseases. Excessive PCD can lead to impaired growth and development, neurodegenerative diseases, and acquired immunodeficiency syndrome (14, 193). On the other hand, indiscrete suppression of apoptosis can result in autoimmune diseases and cancer (62, 101). Tissue injury resulting from hypoxic-ischemic, toxic, and thermal insults can result in both pathological cell death (necrosis) and PCD (apoptosis). Unlike apoptosis that occurs in normal and disease states, necrosis is induced only when cells or tissues are exposed to severe and acute injury (60, 97, 149). The molecular pathways leading to the different modes of cell death in various human diseases and clinical conditions and their regulation have been under intense scrutiny during the past two decades. Dissecting and discriminating the roles of key molecules in various cell death programs are crucial as they are attractive targets for therapeutic intervention.

	MODES OF CELL DEATH

TOP ABSTRACT INTRODUCTION MODES OF CELL DEATH MORPHOLOGICAL, BIOCHEMICAL, AND... MORPHOLOGY OF APOPTOSIS MORPHOLOGY OF NECROSIS ENDONUCLEASES AND DNA... MOLECULAR MECHANISMS OF... APOPTOSIS-BASED THERAPEUTIC... MOLECULAR MECHANISMS OF... CELL DEATH IN ISCHEMIC... MECHANISMS OF NECROSIS IN... MOLECULAR MECHANISMS OF... DEPLETION OF GTP DETECTION OF APOPTOSIS IN... CONCLUDING REMARKS REFERENCES

A distinction between the morphology of cells undergoing physiological cell death and pathological cell death was observed more than 50 years ago (67). However, it was not until the seminal report by Kerr et al. (101) in 1972 that cytologists and pathologists adopted the term "apoptosis" for a morphologically distinct form of cell death. The perpetual flow of information in the past two decades defining the effector and regulatory pathways that result in apoptosis has raised further interest in the role of this mode of cell death in various diseases and pathological conditions, including ischemia-reperfusion injury (IRI) of the heart, brain, and the kidney.

Apoptosis is highly coordinated and is generally thought to be mediated by active intrinsic mechanisms, although extrinsic factors can contribute (16, 30, 205). Apoptosis is genetically controlled and is defined by cytoplasmic and nuclear shrinkage, chromatin margination and fragmentation, and breakdown of the cell into multiple spherical bodies that retain membrane integrity (25, 101, 138, 246). The factors contributing to necrosis are mostly extrinsic in nature, such as osmotic, thermal, toxic, hypoxic-ischemic, and traumatic insults (25, 60). Necrosis is characterized by progressive loss of cytoplasmic membrane integrity, rapid influx of Na⁺, Ca²⁺, and water, resulting in cytoplasmic swelling and nuclear pyknosis (9, 18, 200, 244). The latter feature leads to cellular fragmentation and release of lysosomal and granular contents into the surrounding extracellular space, with subsequent inflammation (138). A recent report indicates that the release of the high-mobility group 1 protein by necrotic cells is involved in promoting the inflammation. Cells deficient for high-mobility group 1 protein showed greatly reduced ability to promote inflammation (201).

Apoptosis and necrosis often occur simultaneously in a wide variety of pathological conditions as well as in cultured cells exposed to physiological activators, physical trauma, or toxins and chemicals (141). The same type of insult can induce either apoptosis or necrosis, but whether one mode of cell death is preferred over the other usually depends on the severity of the insult and the idiosyncrasy of the target cell (9, 25, 128, 141). The perception that apoptosis and necrosis are functionally opposed forms of cell death is fading, and a consensus has developed that both forms of cell death constitute two extremes of a continuum.

	MORPHOLOGICAL, BIOCHEMICAL, AND MOLECULAR MARKERS OF APOPTOSIS AND NECROSIS

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In settings of acute injury such as IRI to the kidney where apoptosis and necrosis coexist (204, 205), discriminating between the two modes of cell death is essential. Despite the recent progress in elucidating the molecular determinants of cell death, a precise histological or biochemical marker to differentiate apoptosis from necrosis has not been identified. Detection of structural alterations using electron microscopy as described originally by Kerr et. al (101) still remains as the reliable criterion to distinguish between the two modes of cell death. It is now agreed upon that a rational combination of at least two techniques should be utilized, one to visualize morphological changes and the second to determine biochemical changes, when the two modes of cell death are asserted.

	MORPHOLOGY OF APOPTOSIS

TOP ABSTRACT INTRODUCTION MODES OF CELL DEATH MORPHOLOGICAL, BIOCHEMICAL, AND... MORPHOLOGY OF APOPTOSIS MORPHOLOGY OF NECROSIS ENDONUCLEASES AND DNA... MOLECULAR MECHANISMS OF... APOPTOSIS-BASED THERAPEUTIC... MOLECULAR MECHANISMS OF... CELL DEATH IN ISCHEMIC... MECHANISMS OF NECROSIS IN... MOLECULAR MECHANISMS OF... DEPLETION OF GTP DETECTION OF APOPTOSIS IN... CONCLUDING REMARKS REFERENCES

In apoptosis, the earliest characteristic change occurs in the nucleus with chromatin condensation, pyknosis, and karyorrhexis (101, 246). The condensed chromatin appears as crescents along the periphery of the nuclear membrane or as spherical bodies within the nucleus. The cytoplasmic condensation instigates the cell to shrink and form numerous vacuoles within the cytoplasm (99, 246). Subsequently, the nuclear and plasma membranes become convoluted, and small masses of condensed chromatin undergo fragmentation along with condensed cytoplasm to form "apoptotic bodies." Apoptotic bodies are plasma membrane bound and often contain functional mitochondria and other organelles (246). The stereotypical morphological changes associated with apoptotic cell death are depicted in Fig. 1. The phosphatidyl serine residues that are normally localized to the inner membrane are relocated to the outside of the cell membrane before its fragmentation. The phosphatidyl serine residues on the apoptotic bodies serve as a signal to the neighboring healthy cells to phagocytose and clear the cellular debris, thus avoiding an inflammatory response (211). In a recent report, Brown et. al (24) provide evidence that homophilic binding between the adhesion receptor CD31 (platelet-endothelial cell adhesion molecule-1) present on leukocytes and macrophages plays a key role in phagocytosis. Both viable cells and dying cells attach to macrophages through these receptors, but the viable cells get detached through an unknown repulsive mechanism. The repulsive signaling is impaired in the dying cells, and they are engulfed (24). It is not known at present whether analogous systems exist in cells other than leukocytes. In vitro, in the absence of phagocytosis, apoptotic bodies ultimately will swell and lyse, and this terminal process of cell death has been termed "secondary necrosis." Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells (243).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Illustration of the various stages of apoptotic cell death. A: depiction of the stereotypical changes including condensation, changes in nuclear structure, and fragmentation of the cell into small apoptotic bodies. In vivo, the apoptotic bodies are phagocytosed by neighboring cells, whereas in vitro they undergo swelling and eventual lysis (secondary necrosis). B: photographs of LLC-PK1 cells undergoing apoptosis at the corresponding stages as shown in A. Apoptosis was induced by overnight exposure of the cells to 50 µM cisplatin. The cells in the first 3 photographs were stained with Hoechst dye, and the cells in the last photograph were stained with acrydine orange and ethidium bromide. In the last photograph, viable cells appear green, whereas the apoptotic cells with intact plasma membrane appear green with yellowish dots representing condensed chromatin; apoptotic cells and bodies that are undergoing secondary necrosis appear bright orange or red due to the plasma membrane damage and entry of ethidium bromide.

	MORPHOLOGY OF NECROSIS

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The morphology of a necrotic cell is very distinct from that of a cell undergoing classic apoptosis, with ultrastructural changes occurring in both the cytoplasm and the nucleus. The main characteristic features are chromatin flocculation, swelling and degeneration of the entire cytoplasm and the mitochondrial matrix, blebbing of the plasma membrane, and eventual shedding of the cytoplasmic contents into the extracellular space (100). Unlike in apoptosis, the chromatin is not packed into discrete membrane-bound particles, but it forms many unevenly textured and irregularly shaped clumps, a feature that is being used for differentiating between the two modes of cell death (224). The mitochondria undergo inner membrane swelling, cristeolysis, and disintegration (112). Polyribosomes are dissociated and dispersed throughout the cytoplasm, giving the cytoplasmic matrix a dense and granular appearance. Dilation and fragmentation of the cisterns of rough endoplasmic reticulum and Golgi apparatus are frequently observed (225).

	ENDONUCLEASES AND DNA FRAGMENTATION IN APOPTOSIS AND NECROSIS

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Analysis of DNA fragmentation by agarose gel electrophoresis is one of the most widely used biochemical markers for cell death. The detection of internucleosomal DNA cleavage (DNA laddering) is considered to be an indicator of apoptosis, whereas the random digestion of DNA resulting in a smear pattern is a marker of necrotic cell death (4, 8, 245). However, recent data from several studies indicate that discriminating between apoptosis and necrosis based on DNA fragmentation pattern is questionable, because both modes of cell death can occur in the absence or presence of DNA fragmentation (49, 192, 229).

The cleavage of double-strand DNA in apoptotic DNA degradation is believed to occur by the activation of endogenous Ca²⁺/Mg²⁺-dependent endonucleases that specifically cleave between nucleosomes to produce DNA fragments that are multiples of ~180 base pairs (35, 229). DNA fragmentation represents a point of no return from the path to cell death, because no more new cellular protein synthesis for cell survival can occur. Although several endonucleases that are involved in apoptosis have been identified and characterized in the past several years, the indispensability of these enzymes for the apoptotic process has been lacking. Two newly identified apoptotic endonucleases [caspase-activated DNAse (CAD) and endonuclease G (Endo G)] with different enzymatic properties appear to be involved in cellular disassembly and cell-autonomous apoptosis (123, 131, 180). The biochemical pathways and the candidate endonucleases involved in DNA degradation are shown in Fig. 2.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Pathways leading to DNA degradation. Mitochondrial derived endonuclease G (Endo G) and nuclear-derived, caspase-activated DNAse (CAD) are 2 known apoptotic endonucleases involved in nuclear DNA degradation. After an apoptotic stimulus, caspase-3 cleaves the inhibitor of CAD (ICAD)/CAD complex and activates CAD to elicit DNA fragmentation in dying cells. Endo G is released from mitochondria in response to various apoptotic stimuli and translocates to nucleus, where it is involved in DNA degradation. The apoptosis-inducing factor (AIF) derived from mitochondria is not a self-nuclease but may participate in DNA degradation, possibly by recruiting other endonucleases. Unlike CAD, activation of Endo G and AIF is independent of caspase activation. It is possible that Endo G and AIF may participate in nuclear DNA degradation when caspase activation is limited.

CAD

Endo G

Apoptosis-inducing factor (AIF) is another molecule involved in chromatin degradation independent of caspase activation. AIF, on its release from mitochondrial intermembrane space, migrates to the nucleus, interacts with DNA (248), and induces partial chromatin condensation and large-fragment (50-kb) DNA fragmentation. However, AIF has no intrinsic endonuclease activity, and AIF-mediated DNA degradation does not require caspase activation (93, 216). It is yet to be determined whether Endo G, CAD, or other endonucleases participate in AIF-mediated DNA degradation (255).

	MOLECULAR MECHANISMS OF APOPTOSIS

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Studies by Horvitz and colleagues (85) first elucidated the existence of a genetically controlled cell death program in which at least three gene products, CED-3, CED-4, and CED-9, participate to cause selective PCD during Caernohabditis elegans development. Subsequent studies in other organisms revealed that several cysteine proteases that share homology to CED-3 are present in mammalian cells. Fourteen such cysteine proteases have been identified so far, and they are identified as caspase-1 to caspase-14 (220, 221).

Molecular Executioners of Apoptosis

Mechanisms of Caspase Activation

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Overview of death-signaling pathways in mammalian cells: The death receptor pathway (left) is initiated by the binding of a ligand (Eg: FasL) to its receptor Fas, which results in the sequential recruitment of FADD and pro-caspase-8. c-FLIP can block the recruitment of pro-caspase-8 to the complex. The proximity of several pro-caspase-8 molecules results in its activation. Caspase-8 can proteolytically activate caspase-3, or it can cleave Bid to its truncated form t-Bid, which binds to Bax and gets integrated into the mitochondrial membrane to release cytochrome c. In response to various cellular stress-induced apoptotic stimuli, the intrinsic mitochondrial pathway is activated. This pathway involves the translocation of proapoptotic molecules such as Bax from the cytosol to the mitochondrial membrane. Bax can release cytochrome c from the mitochondria into the cytosol. Cytochrome c associates with Apaf-1 and caspase-9 to form the apoptosome and subsequent activation of caspase-3. Mitochondria also release AIF and Endo G, which may exert their effects on the nuclei. Mitochondria released Smac/Diablo and Omi/HtrA2 sequesters inhibitors of apoptosis (IAPs) to prevent them from inhibiting caspase-3. BNIP3 is a Bcl2 family member that is translocated and integrated into the mitochondria. Unlike other Bcl2 family members, BNIP3 can induce necrotic cell death in response to death stimuli. Activation of poly (ADP-ribose) polymerase (PARP) leads to NAD+ depletion and may induce mitochondrial depolarization to release AIF. ROS, reactive oxygen species.

Signaling Through Death Receptors

c-FLIP is a naturally occurring dominant negative antagonist of death receptor-mediated caspase-8 activation and contains two DEDs and a defective caspase-like domain. c-FLIP can associate with DEDs of FADD and caspase-8, thus interfering with the recruitment of caspase-8 to FADD (88, 226). Mouse embryonic fibroblasts derived from caspase-8-deficient mice are resistant to Fas, TNF-R1, or DR3 stimulation but susceptible to agents that utilize the mitochondrial death pathway (232). This further demonstrates the crucial role played by caspase-8 in transducing signals downstream of the death receptors.

Mitochondria and Bcl2 Family as Regulators of Cell Death

The ever-growing mammalian Bcl-2 family of apoptotic regulators shares homology with the C. elegans antiapoptotic molecule CED-9 (32, 106). Based on their structure and functional similarities, Bcl2 family members are divided into the proapoptotic (Bax, Bak, and Bok) and antiapoptotic (Bcl2, BclX_L, Bcl-w, Mcl-1, and A1) groups (92). A third class of death effector molecules sharing homology only to the Bcl-2 homology-3 (BH3) domain can activate proapoptotic Bcl2 family members or inactivate antiapoptotic members (86, 213). The family of BH3-only proteins include Bin, Bid, Bad, Bik, BNIP3, Noxa, Puma, and Hrk (86, 162, 213). Pro- and antiapoptotic members of the Bcl-2 family can homodimerize or heterodimerize, thus forming a large number of combinations within a cell. Heterodimerization between a proapoptotic member and an antiapoptotic member can nullify the functions of each (191). The outcome of a cell that received an apoptotic stimulus is thought to depend partly on the ratio of the death promoter to the death suppressor (1, 2). The precise mechanisms by which the Bcl-2 family members modulate apoptosis are still not completely elucidated, but their key functions revolve around the release of proapoptotic factors, especially cytochrome c from the mitochondrial intermembrane compartment into the cytosol (168, 217).

Several models have been proposed to explain how Bcl-2 family members can cause the exit of large molecules such as cytochrome c from the mitochondria. It is suggested that 1) Bcl-2 proteins may insert themselves into the outer mitochondrial membrane, where they could form channels that allow the passage of molecules (191); 2) Bcl-2 family members may interact with other mitochondrial membrane proteins (e.g., the voltage-dependent ion channel or VDAC) to form large-pore channels (191, 209, 227, 228); and 3) Bcl-2 family members may alter mitochondrial membrane permeability, causing mitochondrial swelling and eventual rupture of the outer membrane, thus releasing intermembrane proteins into the cytosol (82). Although evidence to support all of the above models is presented by various investigators, further studies are needed to resolve this crucial issue.

The release of cytochrome c by mitochondria is almost a universal feature found in response to various intracellular stimuli, including DNA damage, glucocorticoids, oxidative injury, and growth factor deprivation, although they may not play a significant role in receptor-mediated apoptosis (3, 109). Cytosolic cytochrome c triggers the formation of the mitochondrial apoptosome, which consists of cytochrome c, apaf-1, and caspase-9 (92, 94). Cytochrome c binds and oligomerizes the adapter protein apaf-1, which recruits pro-caspase-9. This causes pro-caspase-9 to autocatalytically activate to form its active form caspase-9 and proteolytically activate caspase-3. As expected, cytochrome c-deficient embryonic stem cells were resistant to UV light and staurosporine-induced apoptosis and partially resistant to apoptotic stimuli from serum deprivation. However, cytochrome c-deficient cells readily underwent apoptosis in response to receptor-mediated apoptotic stimuli (92, 94).

In addition to cytochrome c, mitochondria release a large number of other polypeptides, including AIF (135), Endo G (see above), second mitochondrial activator of caspases (Smac/DIABLO) (52), HtrA2/Omi (124), and pro-caspases-2, -3, and -9 from the intermembrane space (134). Smac/DIABLO and HtrA2/Omi promote apoptotic cell death by inhibiting a set of proteins termed inhibitors of apoptosis (IAPs) (52, 124). IAPs (e.g., c-IAP1, c-IAP2, X-linked IAP, Survivin) can bind to caspases and block cell death induced by a variety of stimuli (207).

AIF is a flavoprotein that translocates from mitochondria to nuclei on apoptotic stimulation and induces caspase-independent chromatin condensation and large-size (50-kb) DNA fragmentation (216). AIF can also induce mitochondrial release of cytochrome c and thus augment the cell death process through the apoptosome pathway (216). Recent studies indicate that heat shock protein 70, which is upregulated in cellular stress (231), exerts its cytoprotective function by binding and inhibiting AIF activity (190).

Thus mitochondria participate in the apoptotic pathways through at least two independent and redundant pathways, one involving the activation of caspases and the other mediated by AIF. Studies utilizing knockout mice for cytochrome c, apaf-1, caspase-9, and AIF together with caspase inhibition studies suggested that AIF-mediated apoptosis is dependent on the initial stimulus (92). AIF-deficient embryonic stem cells, when cotreated with caspase inhibitors, were protected from apoptosis induced by the oxidative stress inducer menadione, whereas it was only partially protected from apoptosis in response to serum withdrawal. The hierarchical nature and the interactions between AIF and the apoptosome in cell death pathways remain largely unknown (92).

Although the extrinsic and intrinsic signals are considered to take two distinct pathways to execute cell death, receptor-initiated cell death can involve the mitochondrial pathway through the BH3-only protein Bid. In hepatocytes, activation of caspase-8 by Fas leads to cleavage of Bid to its active form t-Bid. t-Bid translocates to mitochondria and associates with Bcl-2-like proteins to disrupt mitochondrial integrity (70, 240). It should be noted that the cross talk between the two pathways is minimal under most conditions. A recent report indicates that cytotoxic stress induced mitochondrial permeability, and release of various apoptogenic factors is mediated by caspase-2 in human fibroblasts transfected with adenoviral oncogene E1A. Small interfering RNA (SiRNA)-mediated silencing of caspase-2 expression prevented cisplatin, etoposide, and UV light-induced apoptosis in these cells. It is argued that in this setting, mitochondria are amplifiers of caspase activity rather than initiators of caspase activation (114).

Mitochondria can also initiate a necrosis-like PCD independently of caspase activation. On stimulation by TNF, mitochondria are shown to produce reactive oxygen species (ROS) that can induce necrotic cell death, and ROS scavengers attenuated the injury (203, 233). An impairment in the cytochrome c or AIF pathway can switch the cell death mode from apoptosis to necrosis. Inhibition of caspases can also switch apoptosis to necrosis once mitochondria are induced. Thus mitochondria play a central role in executing different modes of cell death (116, 118, 119). In addition to mitochondria, several other cellular organelles may participate in apoptotic cell death, and the details are reviewed elsewhere (63).

	APOPTOSIS-BASED THERAPEUTIC AGENTS

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The participation of an ever-growing number of molecules in the apoptotic machinery offers a plethora of opportunities for apoptosis modulation (81). Various strategies that are being developed include the use of antisense oligonucleotides, recombinant proteins, small molecules to disrupt protein-protein interactions, and caspase inhibitors (163, 194).

An antisense oligonucleotide (G-3139) that targets the first six codons of the Bcl-2 open reading frame is shown to downregulate its mRNA. Continuous infusion of G-3139 markedly reduced tumor growth of Merckel cell carcinomas that were xenografted into SCID mice. G-3139 is presently in clinical trials for malignant melanoma and other forms of human cancers. Antisense strategies are also being attempted to modulate the expression of Bcl-XL, c-FLIP, and survivin (163).

Caspase inhibition using active site mimetic peptide ketones, such as fmk [benzyloxycarbonyl (z)-VAD-fluromethylketone], cmk (z-YVAD-fmk/chloromethylketone), z-DEVD-fmk/cmk, and z-D-cmk, have provided valuable information regarding their use as apoptotic inhibitors (66). Caspase inhibition has shown remarkable efficacy in inhibiting apoptotic cell death in different models of IRI, including cerebral, cardiac, and kidney (33, 43, 58). Cell-permeable specific caspase inhibitors are being developed by various pharmaceutical companies and will undoubtedly be more viable in treating acute injuries such as IRI, transplantation, and cerebral stroke (163).

	MOLECULAR MECHANISMS OF NECROSIS

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Necrosis is the prominent mode of cell death that occurs in various neurodegenerative conditions and as a consequence to ischemic injury in various organs including the brain and heart. Even though great progress has been made in the last decade in understanding the molecular mechanisms of apoptosis, the biochemical pathways leading to necrotic cell death remain poorly understood (141). Necrosis is long thought to be a "passive" process occurring as a consequence of acute ATP depletion. Several ATP-dependent ion channels become ineffective, leading to ion dyshomeostasis, disruption of the actin cytoskeleton, cell swelling, membrane blebbing, and eventual collapse of the cell (9, 137, 169). Recent reports suggest that in addition to the passive mechanisms, "active" mechanisms may also participate in the necrotic process.

Na+ Overloading

Ca²+ Accumulation

Recently, two novel Ca²⁺-permeable cation channels belonging to the long transient receptor potential (TRP) channel family, LTRPC2 and LTRPC7, are found to be activated by a disruption of the redox (oxidation-reduction) status (79, 160, 182). LTRPC7 is an intracellular ligand-gated ion channel that can permeate both Ca²⁺ and Mg²⁺ and is suppressed by higher Mg²⁺-ATP concentration. In ischemic conditions, the levels of ATP-bound Mg²⁺ fall, which may activate inward and outward currents through LTRPC7 (160). LTRPC2 is a NSCC that is permeant to both Na⁺ and Ca²⁺. It is activated by binding of ADP-ribose and increased concentration of arachidonic acid and Ca²⁺ but suppressed by high Na⁺ levels. LTRPC2 is activated by micromolar levels of H₂O₂ and agents that produce ROS or reactive nitrogen species, thus representing an intrinsic mechanism that mediates Ca²⁺ and Na⁺ overload in response to changes in redox status (79, 182).

Mild oxidative stress such as that induced by moderate levels of H₂O₂ can increase cytoplasmic Ca²⁺ levels by its release from internal stores such as the endoplasmic reticulum (ER). This process is mediated through protein kinase C and ionositol triphosphate (InsP₃). A recent study demonstrated that in C. elegans, the Ca²⁺-binding proteins calreticulin and calnexin are essential for stimulating necrosis by stresses not involving Ca²⁺ influx across the plasma membrane (247).

The Mitochondrial Connection

One of the major factors that determine whether a cell undergoes apoptosis or necrosis is the level of intracellular ATP. Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that adds ADP-ribose polymers to various proteins and to itself when activated by DNA strand breaks (143). PARP activation is thought to exacerbate ATP depletion and induce necrotic cell death (72, 184). Overactivation of PARP after a cellular injury can result in consumption of its substrate -NAD⁺. In an effort to resynthesize NAD⁺, ATP is massively depleted and the cells die from lack of energy (72, 184). PARP inhibition protected cells from necrotic cell death in neuronal ischemia, myocardial ischemia, and renal ischemia (113, 133, 140). PARP-deficient mice are protected against neuronal and myocardial ischemic injury and diabetic pancreatic damage (56, 183, 257), but they are susceptible to apoptotic cell death elicited by TNF- and Fas in hepatocytes (117) and -irradiation. Fibroblasts from PARP / mice are protected from ATP depletion and necrotic cell death but not from apoptotic cell death (72). A recent study in L929 fibrosarcoma cells showed that ligation of CD95 induces apoptosis, whereas TNF elicits necrosis in the cells. Further investigation into the mechanism of this discrepancy showed that TNF induces PARP activation, leading to ATP depletion and necrosis. In contrast, CD95 ligation cause PARP cleavage, thereby maintaining ATP levels, and the cells die by apoptosis (136).

A recent report by Yu et. al (249), however, indicates that NAD⁺ depletion and subsequent energy depletion may not be the cause of cell death after PARP activation. Instead, NAD⁺ decrement may act only as a signal for AIF translocation from mitochondria to cytosol and to nucleus. AIF initiates nuclear condensation and subsequent chromatin fragmentation, culminating in cellular demise.

	CELL DEATH IN ISCHEMIC ACUTE RENAL FAILURE

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A reduction in the glomerular filtration rate (GFR) is the primary change in renal function caused by ARF in humans and experimental animal models (53). Pathophysiological mechanisms involved in the decline in GFR can be attributed to persistent vasoconstriction due to an imbalance between vasoconstrictive and vasodilatory mediators (36, 37); vascular obstruction caused by endothelial-leukocyte interactions (31, 187); tubuloglomerular feedback in response to increased solute delivery to the macula densa (104, 142); tubular obstruction caused by detachment of tubular epithelial cells from the basement membrane and back-leak of glomerular filtrate as a consequence of disruption of the epithelial cell layer (5, 20, 21, 65).

Vascular Dysfunction in Renal Ischemia

Tubular Dysfunction in Renal Ischemia

Irreversible cell death induced by ischemic, toxic, and obstructive ARF was long considered to be of the necrotic type, but recent data from several laboratories indicate that both apoptosis and necrosis can occur simultaneously in these forms of ARF in humans and experimental animal models (46). The relative contribution of the two mechanisms to the initial cell loss depends on the severity of the injury and the cell type (127).

	MECHANISMS OF NECROSIS IN RENAL ISCHEMIA

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The molecular pathways adopted by the renal tissue that lead to necrotic cell death after an ischemic episode are not fully understood and are now obscured even more by attempts to explore the contribution of apoptosis to renal injury. Hypoxia resulting from decreased blood flow leads to a variety of secondary effects, including a breakdown in cellular energy metabolism, endothelial and epithelial cell dysfunction, cell swelling, generation of ROS, increase in free cytosolic Ca²⁺, and activation of phospholipases, proteases, and endonucleases (20, 197, 229). Recent evidence indicates that active mechanisms such as activation of PARP play important roles in necrotic cell death after renal ischemia (140). A hypothetical scheme of biochemical events that may engage in positive-feedback loops to accelerate cellular disintegration culminating in necrotic cell death is shown in Fig. 4.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Hypothetical sequence of various biochemical events eliciting necrotic cell death. The acute depletion of ATP associated with ischemic-hypoxic injury is exacerbated by mitochondrial dysfunction and activation of various ATP-dependent ion channels, which further depletes ATP by a feedback mechanism. An increase in cytosolic Ca2+ and ROS triggers various converging biochemical pathways, leading to activation of cellular degradative enzymes and ultimate cellular disintegration.

ATP Depletion

Na+ Influx and "Oncosis"

Increased Cytosolic Ca²+ Concentration

Proteases

Meprin (metallopeptidase from renal tissue) is a zinc-dependent metalloendopeptidase that is present in the brush-border membrane of renal proximal tubular epithelial cells, accounting for ~5% of the total tubular protein (223). Meprin is localized to the apical brush border (29, 237). After renal tubular epithelial cell injury, it translocates to the basement membrane and cleaves one of the basement membrane components, nidogen. Mouse strains expressing lower levels of meprin are less susceptible to ischemic injury compared with those expressing normal levels (223). Exogenously added meprin is cytotoxic to renal PTC, and inhibition of meprin provided both histological and functional renal protection after ischemic injury (29, 237).

Phospholipases

Endonucleases

ROS

PARP

Inducible Nitric Oxide Synthase and Osteopontin

Protein Kinases

SAPK/JNK is a member of the MAPK family. SAPK transduces signals to the nucleus in response to cellular stresses such as inflammatory cytokines, ischemia, reversible ATP depletion, heat shock, and genotoxic stress. SAPK activity is markedly increased in the proximal and distal tubules after IRI (110, 178). Inhibition of SAPK activity during ischemia ameliorates renal failure (48). ERKs are another class of the MAPK family involved in mitogenic response and cellular differentiation. ERK is also activated post-renal injury, but its expression is localized to thick ascending limbs in the inner stripe. Studies in several cellular systems have suggested that JNK activation can be modulated by the coexpression of ERKs. It is suggested that the activation of the ERKs in distal tubules during ischemic insult may protect the cells from the injurious effects of JNK activation (47).

	MOLECULAR MECHANISMS OF APOPTOSIS IN RENAL ISCHEMIA

TOP ABSTRACT INTRODUCTION MODES OF CELL DEATH MORPHOLOGICAL, BIOCHEMICAL, AND... MORPHOLOGY OF APOPTOSIS MORPHOLOGY OF NECROSIS ENDONUCLEASES AND DNA... MOLECULAR MECHANISMS OF... APOPTOSIS-BASED THERAPEUTIC... MOLECULAR MECHANISMS OF... CELL DEATH IN ISCHEMIC... MECHANISMS OF NECROSIS IN... MOLECULAR MECHANISMS OF... DEPLETION OF GTP DETECTION OF APOPTOSIS IN... CONCLUDING REMARKS REFERENCES

Apoptotic cell death has been documented in experimental animal models and humans post-renal ischemia, and inhibition of apoptotic cell death is shown to ameliorate the injury and inflammation (42, 43). Several factors that can induce necrotic cell death, including growth factor deprivation, loss of cell-cell and cell-matrix interactions, cytotoxic stimuli, and cell death receptor activation, are also suggested to trigger apoptotic cell death in renal ischemia (126). The severity of the injury caused by these factors and the degree of cellular ATP and/or GTP depletion play crucial roles in determining the mode of cell death (126). A severe depletion of ATP favors necrotic cell death whereas GTP depletion i