An Evaluation of Renal Tubular DNA Laddering in Response to Oxygen Deprivation and Oxidant Injury1
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An Evaluation of Renal Tubular DNA Laddering in Response to Oxygen Deprivation and Oxidant Injury1 M. Iwata, D. Myerson, B. Torok-Storb, and R.A. Zager cols. However, neither protocol induced ethidium M. Iwata, B. Torok-Storb, R.A. Zager, Department of bromide- or tdt-detectable DNA laddering. It was Medicine, University of Washington and the Fred concluded that: ( 1) minimal DNA laddering develops Hutchinson Cancer Research Center, Seattle, WA postischemia, and this change is reliably detected only by the tdt method; (2) it correlates with the D. Myerson, Department of Pathology, University of Washington and the Fred Hutchinson Cancer Re- morphologic expression of tubular necrosis, not ap- search Center, Seathe, WA optosis; and (3) in vitro oxidative- and energy deple- tion-medlated proximal tubular cell death can be (J. Am. Soc. Nephrol. 1994; 5:1307-1313) dissociated from DNA ladder formation. Key Words: Apoptosis. endonuclease. hydrogen peroxide. ABSTRACT ischemia. hypoxia It has recently been suggested that endonuclease activation and/or apoptosis, possibly triggered by poptosis is an internally regulated program by oxidant stress, are Important pathogenetic mecha- A which cells can be eliminated from tissues in a nisms in oxygen deprivation/reoxygenatlon-induced controlled fashion in response to a variety of stimuli proximal tubular cell death. To explore this possibility, (reviewed in References 1-5). It has been widely pro- DNA laddering,” a characteristic feature of these posed as a mechanism for the elimination of cells processes, was sought in: ( 1) postlschemic rat kidneys during development, for the removal of senescent (25- or 40-mm arterial clamping; 0, 1, 4, 8, 24, and 48 h cells, and for the control of excessive proliferation and 6 days reflow); (2) posthypoxic isolated rat prox- during tissue regeneration. It may also be Induced by imal tubular segments and (3) cultured human kid- a variety of disease processes (e.g. , neurodegenerative ney proximal tubular cells (HK-2) subjected either to disorders, radiochemotherapy, oxidative stress), by energy depletion plus Ca2 overload (antimycin A selected regulatory hormones (e.g. , glucocorticoid ad- plus 2-deoxyglucose plus Ca2 ionophore A23187), or dition to lymphocytes), or by the withdrawal of specific trophins (e.g. , interleukins, estrogen. testosterone) to H202-induced cell death. DNA was subsequently (1-6). Irrespective of the stimulus, strikingly similar extracted, electrophoresed through agarose gels, morphologic changes result, most notably chromatin and visualized with ethidium bromide or Southern condensation and nucleolar degeneration, nuclear blotting. To maximize ladder detection, DNA samples compaction, and decreased cell volume (3). Subse- were also end-labeled with 32P dideoxyadenosine quently, the involved cells fragment into apoptotic triphosphate with terminal deoxynucleotidyl trans- bodies” that are rapidly eliminated, either by phago- ferase (tat), followed by electrophoresis. None of the cytosis or by in situ degeneration. From a pathogenetic postischemlc DNA samples demonstrated any lad- standpoint, apoptosis is believed to differ markedly dering by either ethidium bromide staining or South- from ischemic on toxic cell death (7): the latter are em analysis (apoptotic lymphocyte DNA was a post- externally, not internally, controlled: they usually af- five control). However, trace laddering was apparent fect large numbers of contiguous cells in a relatively nonspecific fashion; and early cell swelling, not con- by the tat technique, commencing at 1 h of reflow, densation, results. peaking at 24 h, and resolving slowly thereafter. This Although the precise mechanisms that mediate ap- finding correlated with the morphologic expression of optosis are poorly defined, a characteristic feature of it tubular necrosis, not apoptosis. Hypoxia/reoxygen- is endonuclease activation, which results In the deg- ation caused proximal tubular segment death (44 to radation of genomic DNA at intennucleosomal linker 64%), and HK-2 cells were slowly killed by both the regions. This process generates 180- to 185-base-pain H2O2 and the energy depletion/Ca24-Ioadlng proto- fragments, which by agarose gel electrophoresis, yield a “laddered” DNA appearance (e.g., References 4 and 1 ReceIved March 25, 1994. Accepted June 15, 1994. 6). Because the morphologic changes of apoptosls are 2 corresponcience to Dr. R.A. Zoger, Fred Hutchinson Cancer Research Center, extremely transient and, thus, are easily missed, DNA 1 124 ColumbIa Street, Seattle, WA 98104. laddening” has become widely accepted as a marker 1046.6673/0506-1307$03.00/0 of apoptotic cell death. Journal of the American SOCiety of Nephrology Copyright C 1994 by the American SOCIetY of Nephrology In 1992, Schumer et aL reported that DNA, Journal of the American Society of Nephrology 1307 Renal Tubular DNA Laddenng extracted from rat kidneys 1 2 to 48 h postischemia ing served as negative controls. As a positive control, (5-, 30-, 45-mm arterial clamping), generated charac- laddered DNA was obtained from 2B4 lymphocytes, which teristic apoptotic ladders, as assessed by agarose gel were rendered apoptotic by exposure to 1 M dexarnethasone electrophoresis with ethidium bromide staining (8). for 24 h (N = two preparations) (12). In an effort to correlate the results with the morphologic On the basis of those observations, it has been sug- appearance ofthe kidneys from which the DNAwas obtained, gested that apoptosis may be triggered by ischemia, coronal kidney slices were taken after each of the reflow potentially contributing to the development of post- periods, they were fixed by immersion in either 10% buffered ischemlc tubular cell death and, hence, acute renal formalin or methyl Carnoy’s, and subsequently, 5-sm sec- failure (ARF) (8,9). To gain support for this intriguing tions were cut and stained with hematoxylin and eosin. They possibility, our laboratory recently used a newly de- were examined by a pathologist (D. Myerson) for the appear- scribed histocytochemical technique (10) to ascertain ance of tubular necrosis and apoptosis (as defined above) whether DNA damage can be observed in situ in without knowledge of the specific experimental protocol that postischemic rat kidneys (1 1). ThIs method uses for- had been undertaken. main-fixed tissues that are incubated with terminal In Vitro 02 Deprivation Injury: Isolated Proximal deoxynucleotidyl transferase (tdt) and biotinylated de- Tubular Segment Experiments oxyunidine, the latter being incorporated into the 3’-OH ends of DNA, exposed with DNA breaks (10). Since the onset of tubular cell death cannot be precisely Subsequently, the deoxyunidlne signal is amplified timed in vivo by morphologic assessments, It was impossible with avidin penoxidase. allowing for its detection by to discern from the in viva experiments whether DNA frag- mentation precedes the loss of cell viability, as would be conventional light microscopy. The results of that expected If It were helping to mediate tubular cell death. To study indicated that DNA breaks can be found within gain possible insights Into this Issue, DNA laddening in postischemic rat kidneys; however, they appeared to response to hypoxic-reoxygenation injury was sought In be limited to those tubular cells that were already isolated rat proximal tubular segments (FF5), because the overtly necrotic, suggesting that the DNA changes loss of cell viability can be precisely timed in this system by were a result, rather than a mediator, of postischemic lactate dehydrogenase (LDH) release. Three sets of FF5, tubular cell death. Furthermore, although DNA prepared as previously described (13). were each divided into breaks were observed in that study, in situ detection three equal aliquots as follows: ( 1) continuous oxygenation with the tdt method does not necessarily mean that (95% 02-5% CO2 incubation for 45 mm); (2) hypoxic injury (95% N2-5% CO2 incubation for 30 raIn): and (3) 30 mm of characteristic apoptotic laddering had resulted. In hypoxia followed by 15 mm ofreoxygenation, each performed other words, the DNA changes could have reflected as previously described (13). At the completion of each nonspecffic DNA degradation, rather than endonucle- incubation, percent LDH release was determined (the ase-mediated ‘apoptotic” death. amount of total tubular LDH released into the PTS bathing As recently suggested by Bonventre (9), the docu- media) (13). Finally, the FF5 suspension was pelleted by mentation of apoptosls, or DNA laddening, in early centrifugation, and the DNA was extracted and analyzed with postischemic renal tissues could substantially alter ethidium bromide-stained gels and the tdt method, as de- our current thinking about the mechanisms of post- scrlbed below. ischemic ARF. Therefore, the goals of this study were Energy Depletion-Induced Cell Death in to confirm the presence of laddered DNA in postis- chemic rat kidneys by a variety of methods and to Cultured Human Proximal Tubular Cells: further explore its pathogenetic significance with Evaluation of DNA Ladder Formation three in vitro models of proximal tubular cell death. To further assess whether DNA fragmentation is an early pathogenetic factor in energy depletion-induced tubular cell METHODS death, additional experiments were performed with a newly described cultured human proximal tubular cell line, HK-2 In Vivo lschemia/Reperfusion Experiments (14). The cells were grown in flasks in keratinocyte-serum Male Sprague Dawley rats (175 to 275 g)