and toxic renal injury. A major advance in the understanding of cell death is the fact that many features of the cell-signalling process, leading to apoptotic forms ...
Nephrol Dial Transplant (2001) 16 wSuppl 6x: 23–26
Apoptosis in acute renal failure Marc E. De Broe Department of Nephrology, University of Antwerp, Belgium
The landmark publication by Kerr et al. in 1972 w1x, describing apoptosis as a new basic phenomenon with wide ranging implications in tissue kinetics, has changed our thinking about cell death (Figure 1). Necrosis and apoptosis differ fundamentally from each other. Unlike necrosis, apoptosis is mediated by an organized, predetermined pathway that can be divided into two stages: the commitment phase and the execution phase. The differences between apoptosis and necrosis are summarized in Table 1. 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. The plasma membrane becomes convoluted and the cells separate into a cluster of membrane segments after containing normal mitochondria and other cellular organelles. The absence of inflammation in apoptosis permits cell death without damage to surrounding allowing development and homeostasis of organs under physiological and pathological conditions. 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 w2x. Ueda and Shah w3–5x correctly pointed out recently that equating DNA fragmentation with apoptosis is problematic: chromatin condensation and DNA fragmentation are regulated by different metabolic pathways, apoptosis can occur without DNA fragmentation and, DNA fragmentation can be seen in necrotic form of cell death. The most frequently used methodology nowadays to visualize and quantify apoptosis is the in situ end labelling of fragmented DNA following proteinase K digestion (TUNEL). In our hands w6,7x and that of others w8x, this TUNEL methodology labels a high proportion of non-apoptotic nuclei and consequently overestimates apoptosis. In addition, apoptosis has been reported to be coupled with the appearance of
Correspondence and offprint requests to: Marc E. De Broe, University of Antwerp, Department of Nephrology-Hypertension, pua University Hospital Antwerp, Wilrijkstraat 10, B-2650 Edegemu Antwerpen, Belgium. #
PCNA as well as significant Brdu incorporation. These results suggest that en route to apoptosis, cells undergo events typical of early cell-cycle traverse by expressing G1 genes and may even experience the late G1uG-phase boundary, as shown by the presence of PCNA before they abort and die w9x. The term apoptosis should only be applied when there are morphological criteria as described in the original paper by Kerr et al. w1x.
Apoptosis in ischaemiaureperfusion and toxic renal injury A major advance in the understanding of cell death is the fact that many features of the cell-signalling process, leading to apoptotic forms of cell death, are shared with those associated with necrotic form of cell death. Lieberthal et al. w10x has shown that the proximal tubule cells subjected to severe ATP depletion die by necrozing whereas moderate ATP depletion results in apoptosis; they also showed that a low dose of cisplatin results in apoptosis whereas high dosage induces necrosis w11x. One of the first in vivo studies demonstrating morphological biochemical and molecular evidence of apoptosis during the reperfusion phase (as early as 12 h) after brief periods of renal ischaemia is from Schumer et al. w12x. Ueda and Shah w13x provided strong evidence for the role of endonuclease activation in DNA damage and cell death in hypoxiaure-oxygenation injury. Of note is the fact that 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. Kaushal et al. w14,15x demonstrated the participation of caspases in hypoxic injury to the renal tubular cells and ischaemiaureperfusion injury to the kidney. In several non-ischaemic causes of acute renal failure, such as endotoxaemia, cisplatin, HgCl2, doxorubicin, cyclosporin, radiocontrast media, apoptosis, have been shown w3x. We induced a selective necrosis of the kidney proximal convoluted tubule by injecting
2001 European Renal Association-European Dialysis and Transplant Association
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M. E. De Broe
Fig. 1. Apoptosis: a basic biologic phenomenon with wide ranging implications in tissue kinetics. Adapted from w1x. Table 1. Cell death Apoptosis
Necrosis
Programmed death of unwanted cells (loss of growth stimuli) Hours (12–18) Individual cells . Intact membrane . Nuclear condensation . Cell shrinkage . Blocking of cell surface . Cell fragmentation . Organelles retain integrity
Accidental, unplanned severe injury (toxins, ischaemia, . . . ) Minutes Adjacent areas . Necrosis, plasma membrane disruption . Cytoplasm and cell volume "
Suicide . Needs energy . Activation of enzymes (endonucleases) . Save removal by phagocytes . No inflammation . Inconspicuous
Murder . Loss metabolic activity . Loss of enzyme . Leakage-inflammation . Lysis . Messy, spectacular
subcutaneously the aminoglycoside gentamicin to rats w16x. After injury, the strongly increased cell proliferation in regenerating necrotic PCT was preceded by an equally important proliferation in the distal tubules of
. Organelles swell
the cortex and outer stripe of the outer medulla in the absence of necrosis but displaying enhanced apoptosis (day 2 after injury). Yet, epithelial vimentin expression, a marker of migrating proliferating cells, was restricted
Apoptosis in acute renal failure
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Fig. 2. Occurrence of apoptosis in cortical proximal and distal tubules during injury and recovery from gentamicin-induced acute renal failure (means"SD). Adapted from w16x.
to regenerating PCT. After full recovery of the PCT, a second burst in proliferative activity occurred in the hypertrophic distal segments most likely in response to functional overload marked by transient PCT necrosis-dysfunction. In the regenerated PCT, an excess cell number was accompanied by increased apoptotic activity (day 10 after injury) (Figure 2). There was a striking parallelism between the occurrence of the first proliferative wave at days 0–4 and the prevalence of apoptotic nuclei in the distal tubules. Apoptosis of programmed cell death is a process that occurs as part of the normal cell turnover and in tissue remodelling and may be increased by several types of injury. Increased apoptosis and cell proliferation have been observed in distal tubules after brief ischaemia without necrosis, and in scar-adjacent tissue in the remnant kidney. Also, during ureteric obstruction, apoptosis is induced primarily in the distal tubule w17x. Caspases are involved in apoptosis associated with toxic acute renal failure, as was recently shown in cisplatin-induced cell death in mouse proximal tubular cells w18x. Evidence for a role of ceramide in apoptosis comes from the following observation: (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. Ueda et al. w19x showed that ceramide is a regulator of endonuclease in renal tubular epithelial cell injury.
Therapeutic implications The inhibition of apoptosis may be undesirable such as the apoptotic death of cells by chemotherapeutic agents with irreparable DNA damage. It is not clear that apoptotic death induced in response to mild ischaemic or toxic injury serves any useful
‘homeostatic role’. The observations we made in a toxic model of acute renal injury seem to support a role of apoptosis in the remodelling of the proliferative regenerating proximal and hyperplastic hypertrophic distal tubule w16x. During the commitment phase, the potent prosurvival effect of anti-apoptotic Bcl-2 family member proteins, the activity of the transcription factor NF&B and the upstream signalling kinase pathways represent the three areas for pharmacological intervention w20x. In addition, during the early execution phase the caspase system can be modulated, using cell permanent inhibitors. They have been shown to prevent tubular cell death in response to ischaemic injury w21x. It is clear that more insights into the events involved in apoptosis are needed before therapeutic agents interfering with this system can be used.
References 1. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257 2. Wylie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980; 284: 555–556 3. Ueda N, Sha SV. Tubular cell damage in acute renal failure – apoptosis, necrosis, or both. Nephrol Dial Transplant 2000; 15: 318–323 4. Ueda N, Walker PD, Hsu S-M, Sha SV. Activation of a 15-kDa endonuclease in hypoxiaureoxygenation injury without morphologic features of apoptosis. Proc Natl Acad Sci USA 1995; 92: 7202–7206 5. Ueda N, Kaushal GP, Shah SV. Apoptotic mechanisms in acute renal failure. Am J Med 2000; 108: 403–415 6. Duymelinck C, Dauwe SEH, De Greef KEJ, Ysebaert DK, Verpooten GA, De Broe ME. TIMP-1 gene expression and PAI-1 antigen after unilateral ureteral obstruction in the adult male rat. Kidney Int 2000; 58: 1186–1201 7. Zhu MQ. Apoptosis and Re-epithelialization in the Rat Kidney After Toxic Injury. PhD thesis, University of Antwerp, 1997 8. Schaper J, Elsa¨sser A, Kostin S. The role of cell death in heart failure. Circulation 1997; 95: 320–323
26 9. Pandey S, Wang E. Cells en route to apoptosis are characterized by the upregulation of c-fos, c-myc, c-jun, cdc2 and RB phophorylation, resembling events of early cell-cycle traverse. J Cell Biochem 1995; 58: 135–150 10. Lieberthal W, Menza SA, Levine JS. Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells. Am J Physiol 1998; 274: F315–327 11. Lieberthal W, Triaca V, Levine J. Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am J Physiol 1996; 270: F700–708 12. Schumer M, Colombel MC, Sawczuk IS et al. Morphologic, biochemical and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 1992; 140: 831–838 13. Ueda N, Shah SV. Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest 1992; 90: 2593–2597 14. Kaushal GP, Ueda N, Shah SV. Role of caspases (ICEuCED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A. Kidney Int 1997; 52: 438–445 15. Kaushal GP, Singh AB, Shah SV. Identification of caspase (ICE-like proteases) gene family in rat kidney and altered
M. E. De Broe
16.
17. 18.
19.
20. 21.
expression in ischemiaureperfusion injury. Am J Physiol 1998; 274: F587–595 Nouwen EJ, Verstrepen WA, Buyssens N, Zhu MQ, De Broe ME. Hyperplasia, hypertrophy and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat. Lab Invest 1994; 70: 479–493 Gobe GC, Axelsen RA. Genesis of renal tubular atrophy in experimental hydronephrosis in the rat – role of apoptosis. Lab Invest 1987; 56; 273–281 Fukuoka K, Takeda M, Kobayashi M et al. Distinct interleukin1b-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells. Life Sci 1998; 62: 1125–1138 Ueda N, Kaushal GP, Hong X, Sha SV. Role of enhanced ceramide generation in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Kidney Int 1998; 54: 399–406 Rana A, Sathyanarayana P, Lieberthal W. Role of apoptosis of renal tubular cells in acute renal failure: therapeutic implications. Apoptosis 2001; 6: 83–102 Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312–1316
