Tubular cell damage in acute renal failure—apoptosis, necrosis, or both

Norishi Ueda and Sudhir V. Shah

Department of Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA

Correspondence and offprint requests to: Sudhir V. Shah MD, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Slot 501, Little Rock, AR 72205, USA

Keywords: acute renal failure; caspases; cell death; ceramide; endonucleases; reactive oxygen metabolites; renal tubular epithelial cells

Introduction

For almost a century, it has been taken as a foregone conclusion that in ischaemic or toxic injury, renal tubular epithelial (RTE) cells die in a catastrophic breakdown of regulated cellular homeostasis, that is necrosis (Figure 1Go). Necrosis is accompanied by massive tissue damage leading to rapid collapse of internal homeostasis of the cell [1]. It is characterized by cell swelling with early loss of plasma membrane integrity, major changes to the organelles, and swelling of the nucleus with flocculation of the chromatin. Affected cells rupture, and the cellular components spill into the surrounding tissue space, evoking an inflammatory response [1,2]. In necrosis, DNA degradation is a later phenomenon, if present, and the chromatin is digested by proteases and endonucleases into a smear pattern instead of a ladder pattern since the proteases destroy the histones and expose the entire length of DNA to the nucleases [2].



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Fig. 1. Morphology and biochemical characteristics of apoptosis and necrosis. (Used with permission from Mosby-Year Book, Inc.).

 
The landmark study by Kerr et al. in 1972 [1] has changed our thinking about cell death and there is an explosion of data indicating that the unreflected dogma of necrosis as sole mechanism of cell death is wrong and incomplete. Kerr et al. in their publication made a distinction between necrosis and apoptosis based on morphological criteria (Figure 1Go and Table 1Go). They adopted the term `apoptosis' from Greek, which describes dropping off or falling off petals from flowers, or leaves from trees. To show the derivation clearly they proposed that the second half of the word be pronounced `ptosis' with the `p' silent, which comes from the same root `to fall', and is already used to describe drooping of the upper eyelid. In apoptosis, the most outstanding morphological and biochemical changes occur in the nucleus in which chromatin rapidly forms dense crescent-shaped aggregates lining the nuclear membrane [13]. Complex invaginations develop in the nuclear membrane, resulting in a segmented nucleus. The plasma membrane becomes convoluted, so that the cell separates into a cluster of membrane-bound segments, `apoptotic bodies', which often contain morphologically normal mitochondria and other cellular organelles. At this stage the change in the cells' surface occurs and apoptotic cells are immediately phagocytized by macrophages or other adjacent viable cells without causing an inflammatory response [1,3]. The absence of inflammation is a crucial feature of apoptosis, and thus it permits cell death without damage to adjacent cells, and is thus advantageous for normal cell turnover, development, and homeostasis of organs under physiological and pathological conditions. In fact, in embryogenesis, events occur at such predicted time points that it is often referred to as programmed cell death, and often erroneously used interchangeably with apoptosis [3].


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Table 1. Characteristics of apoptosis and necrosis
 
In apoptosis, endogenous endonuclease activation has been shown to result in the cleavage of host chromatin into oligonucleosome-length DNA fragments (~200 bp), considered a characteristic biochemical marker for apoptosis [4]. In fact, in a large number of studies published in the literature, the presence of DNA fragmentation has been equated with apoptosis. This is, at least in part, attributable to the fact that in earlier studies DNA fragmentation was invariably associated with response to stimuli that led to apoptosis. In fact, this led to the earlier belief that both chromatin condensation and DNA fragmentation were a result of endonuclease activation. Several recent observations make equating DNA fragmentation with apoptosis problematic: chromatin condensation and DNA fragmentation are regulated by different metabolic pathways (cited in [5]), apoptosis can occur without DNA fragmentation [5], and, as described below, DNA fragmentation can be seen in necrotic form of cell death. In our view the term apoptosis should only be applied when there are morphological criteria as described in the original paper by Kerr et al. [1].

One of the major advances in our understanding of cell death has been the recognition that the pathways traditionally associated with apoptosis may be very critical in the form of cell injury associated with necrosis. It is likely that many features of the cell signalling process leading to apoptotic form of cell death are shared with those associated with necrotic form of cell death. The pathway that is followed by the cell is dependent on both nature and severity of insults, evolving from apoptotic to necrotic form of cell death (Figure 2Go). Thus, it is now recognized that the same insult in a mild form can lead to apoptosis and when severe can lead to necrosis. As an example, the recent study by Lieberthal et al. [6] has shown that the proximal tubule cells subjected to severe ATP depletion die by necrosis, whereas moderate ATP depletion results in apoptosis. Investigators from the same laboratory also showed that a low dose of cisplatin results in apoptosis whereas high dosage induces necrosis [7]. Other reviews on the role of apoptosis in renal tubular cell injury have also been published [8].



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Fig. 2. Outcome of renal tubular epithelial (RTE) cells following noxious insults. The pathway that is followed by the cell is likely to be dependent on both nature and severity of insults. The same insult in a mild form can lead to apoptosis and when severe can lead to necrosis. Cells exposed to transient or mild insult may be able to recover. It appears likely that the cascades that lead to apoptotic and necrotic form of cell death are activated almost simultaneously and that there are some common pathways that are shared and regulated in the two modes of cell death. (Used with permission from S. Karger.).

 
Ischaemic acute renal failure

Our own interest in the role of endonucleases in RTE cell injury began with our attempts to understand the biochemical and cellular mechanisms of oxidant injury. It was generally accepted that hydrogen peroxide gets to the DNA, and in the presence of iron in the DNA results in the site-specific generation of hydroxyl radical which causes the DNA damage. We were able to show that, in fact in hydrogen peroxide-induced injury to RTE cells, endonuclease activation occurs as an early event leading to DNA fragmentation and that endonuclease inhibitors prevented hydrogen peroxide-induced DNA strand breaks, DNA fragmentation, and cell death [9]. Several recent studies support the idea that reactive oxygen metabolites (ROM) may be important in endonuclease activation [10]. For example, low doses of hydrogen peroxide or agents that increase ROM induce apoptosis, and high doses induce necrosis, indicating that the severity of insult is the determinant of cell death [11]. Based on the demonstration of the role of endonucleases in oxidant injury, we surmised that endonuclease activation may be important in those forms of acute renal tubular injury where ROM have been implicated [12,13].

Our data led us to consider whether endonuclease activation may be important in hypoxia/reoxygenation injury, generally considered to lead to necrotic form of cell death. We demonstrated that hypoxia/reoxygenation to freshly isolated rat proximal tubules resulted in DNA strand breaks and nuclear DNA fragmentation that preceded cell death [5]. Hypoxia/reoxygenation resulted in an increase in DNA degrading activity with an apparent molecular mass of 15 kDa. Endonuclease inhibitors provided complete protection against DNA damage induced by hypoxia/reoxygenation and partial but significant protection against cell death. Taken together our data provide strong evidence for a role of endonuclease activation in DNA damage and cell death in hypoxia/reoxygenation injury. It is important to note that despite unequivocal evidence of endonuclease activation, light and electron microscopy did not observe the morphological features of apoptosis, including chromatin condensation. This is consistent with recent studies indicating that chromatin condensation and DNA fragmentation may be triggered through separate metabolic pathways (cited in [5]). Several studies have shown apoptosis in renal tubule cells including LLC-PK1 cells [14], MDCK cells [14,15], and mouse proximal tubule cells subjected to chemical hypoxia as well as rat kidney [6] proximal tubules subjected to ATP depletion [16]. In general the in vitro studies have shown apoptosis and DNA fragmentation in renal tubular cells during hypoxia as well as reoxygenation.

One of the first in vivo studies to document the occurrence of apoptosis in ischaemic acute renal failure was a study by Schumer et al. [17], where apoptosis has been observed in rat kidney cortex 12 h after reperfusion. Much of the evidence for the role of apoptotic mechanisms in RTE cell injury relates to the demonstration of endonuclease activation resulting in oligonucleosome-length DNA fragmentation.

The study by Miller et al. [18] showed that isolated perfused rat kidneys subjected to hypoxia develop DNA strand breaks along the distal tubule, as detected by a histochemical technique based on terminal deoxynucleotidyl transferase (Table 2Go). Iwata et al. [19] reported DNA fragmentation after in vivo ischaemia/reperfusion injury associated with morphological features of necrosis rather than apoptosis. Based on the information in the literature, the in vivo studies of ischaemia/reperfusion so far demonstrate apoptosis only during the reperfusion period.


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Table 2. Evidence for apoptosis and necrosis in in vitro and in vivo models of acute renal failure
 
Other causes of acute renal failure

Endotoxaemia is one of the important causes of acute renal failure, and apoptosis has been shown to be associated with this model of renal failure. Recent in vitro studies showed that apoptosis occurs in renal tubule cells in response to Escherichia coli Shiga-like toxins [20,21]. It was shown that TNF-{alpha} enhances endotoxin-induced apoptosis in renal tubular cells [20,22]. Similar observation has been made in in vivo studies in which LPS could induce apoptosis in the kidney [23,24].

Agents used for diagnosis and treatment can trigger apoptosis in renal tubule cells (Table 2Go). They include chemotherapeutic agents such as cisplatin [7,25,26] and doxorubicin [27], cyclosporin A [28], and radiocontrast because of its hypertonicity [29,30]. Antibiotics may possess a potential to induce apoptosis. An experimental study showed that gentamicin induced apoptosis in renal distal tubules during the acute phase and in proximal tubules during the recovery phase [31]. The severity of insults is a determinant for cells' decision to either undergo apoptosis or necrosis, because the lower dose of agents such as cisplatin [7] and HgCl2 [32] can induce apoptosis, whereas the higher dose causes necrosis.

Apoptosis has been shown in allografted kidneys from patients with acute or chronic rejection [3335]. It was also shown that DNA fragmentation in situ occurs during reperfusion in human renal allografts from cadaveric donors but not living-related donors [36]. Hydronephrosis due to ureteral obstruction has also been shown to induce apoptosis or DNA fragmentation in the kidney [3740].

If endonuclease activation, traditionally regarded as a biochemical hallmark of apoptosis, is important in hypoxia/reoxygenation injury, we then asked whether the mediators that have been traditionally associated with apoptosis are applicable to hypoxic injury.

Role of caspases in RTE cell injury

Caspases (ICE/CED3 family of proteases) are a family of cell death proteases that play a key role in the execution of apoptosis. The term caspases signifies two distinct properties of these enzymes in which `c' refers to the cysteine proteases and `aspase' denotes their specificity to cleave after aspartic acid. The first caspase was discovered in 1993, when it was shown that the cell death gene, CED3, in Caenorhabditis elegans has sequence homology to caspase-1, which was then called the interleukin-1ß-converting enzyme (ICE). So far, 14 members of caspase family have been identified from mammalian cells [41]. Overexpression of executioner and initiator caspases in transfected cells results in DNA fragmentation and cell death in a variety of transfected mammalian cell lines. At least 40 different protein substrates for caspases have been recognized, which include DNA repair enzymes, DNA fragmentation factor responsible for internucleosomal DNA cleavage, nuclear structural proteins, cytoskeleton proteins, and caspases themselves. It is assumed that these or more unknown substrates cleaved by caspases are responsible for the changes that occur during the process of apoptosis.

Currently there is limited information on the role of caspases in ischaemic renal tubular cell injury. In our previous studies we have demonstrated the participation of caspases in hypoxic injury [42] to renal tubular cells and ischaemia/reperfusion injury to the kidney [43]. We have shown that chemical hypoxia with antimycin A results in increased caspase activity that precedes DNA damage and cell death and that the inhibition of caspases prevented hypoxia-induced DNA damage and cell death in LLC-PK1 cells [42]. Partial ATP depletion in MDCK cells induced by chemical hypoxia also has been shown to result in apoptosis with marked increase in activation of caspase-8 and the caspase inhibitors rescued apoptosis [44]. In a related study, activation of caspase-3 during hypoxia or ATP depletion has been shown to be accompanied by cytochrome c release from mitochondria [16]. Using an in vivo model of ischaemia/reperfusion injury we have demonstrated activation as well as increased expression of caspase-1 and caspase-3 mRNA [43].

Caspases are involved in apoptosis associated with toxic acute renal failure. Activation of caspase-3 was recently shown to be accompanied in cisplatin-induced cell death in mouse proximal tubular cells, and overexpression of crmA, a cowpox virus gene known to inhibit caspases, provided marked protection against cisplatin-induced apoptosis in mouse proximal tubular cells [25,26]. Taken together these data imply that caspases function as an executioner of apoptosis induced by toxic acute renal failure.

Role of ceramide in RTE cell injury

The major role of membrane lipids has been thought to be a structural barrier for cell permeability as well as a matrix for the association of membrane proteins. However, it is now recognized that the products formed by hydrolysis of membrane phospholipids can serve as key molecules in cell signalling pathway [45]. Recently ceramide, a metabolite of sphingolipids, has been implicated to play an important role in cell signalling pathway involved in apoptosis [45]. Two major pathways generate ceramide: condensation of sphingosine or sphinganine and fatty acyl-CoA by ceramide synthase or hydrolysis of sphingomyelin by sphingomyelinases. Evidence for a role of ceramide in apoptosis comes from the following observations: (i) exogenous ceramide induces apoptosis; (ii) increased ceramide level is seen in apoptotic cells in response to a variety of stimuli; (iii) cells deficient in ceramide or low response of ceramide production are resistant to apoptosis; and (iv) the inhibition of ceramide rescues apoptosis.

Recent studies by Zager et al. [46] showed increased ceramide level during reperfusion of ischaemic mouse kidney and in hypoxic RTE cells accompanied by a decrease in sphingomyelinase activity. We postulated that ceramide may play an important role as a modulator of endonuclease in RTE cell injury. We have shown that subjecting RTE cells to hypoxia results in a rapid increase in ceramide generation, and that inhibition of ceramide synthase prevents hypoxia-induced ceramide generation, DNA strand breaks, DNA fragmentation, and cell death [47]. In addition, exogenous ceramide results in DNA damage and cell death similar to the effect of hypoxia. These data do implicate that ceramide is a regulator of endonuclease in RTE cell injury. In addition, ceramide has been shown to be increased by ROM and result in activation of caspases [48]. Thus it appears likely that these mediators sequentially or in concert regulate endonuclease resulting in RTE cell injury. Although a number of cellular targets for ceramide have been identified [45], the precise mechanisms by which ceramide regulates endonucleases remain to be elucidated.

Conclusion

Along with the rapid increase in the number of publications and interest in the study of cell death, has come some confusion of the terminology. In our view, the term apoptosis should only be applied when there are morphological criteria as described in the original paper [1]. The DNA fragmentation frequently used as the sole criteria for apoptosis is probably misleading because this feature traditionally regarded as the biochemical marker for apoptosis is, in fact, seen in many forms of cell injury that lead to necrotic form of cell death and do not have morphological features of apoptosis. In addition, cell signalling for nuclear condensation and DNA fragmentation is now known to be regulated by different metabolic pathways. It is likely that many features of cell signalling process leading to apoptotic form of cell death are shared with those associated with necrotic form of cell death. The pathway that is followed by the cell is dependent on both nature and severity of insults, evolving from apoptotic to necrotic form of cell death. A better understanding for the mechanisms by which endonucleases are regulated would provide new therapeutic opportunities of acute renal failure.

Acknowledgments

This work was supported in part by the Department of Defense, Office of the Navy, N00014-95-1-0583, the National Institutes of Health, R01 DK47990, and the VA Merit Review Award of the Central Arkansas Veterans Healthcare System.

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