An evaluation of renal tubular DNA laddering in response to oxygen deprivation and oxidant injury.

Abstract

It has recently been suggested that endonuclease activation and/or apoptosis, possibly triggered by oxidant stress, are important pathogenetic mechanisms in oxygen deprivation/reoxygenation-induced proximal tubular cell death. To explore this possibility, DNA "laddering," a characteristic feature of these processes, was sought in: (1) postischemic rat kidneys (25- or 40-min arterial clamping; 0, 1, 4, 8, 24, and 48 h and 6 days reflow); (2) posthypoxic isolate rat proximal tubular segments and (3) cultured human kidney proximal tubular cells (HK-2) subjected either to energy depletion plus Ca2+ overload (antimycin A plus 2-deoxyglucose plus Ca2+ ionophore A23187), or to H2O2-induced cell death. DNA was subsequently extracted, electrophoresed through agarose gels, and visualized with ethidium bromide or Southern blotting. To maximize ladder detection, DNA samples were also end-labeled with 32P dideoxyadenosine triphosphate with terminal deoxynucleotidyl transferase (tdt), followed by electrophoresis. None of the postischemic DNA samples demonstrated any laddering by either ethidium bromide staining or Southern analysis (apoptotic lymphocyte DNA was a positive control). However, trace laddering was apparent by the tat technique, commencing at 1 h of reflow, peaking at 24 h, and resolving slowly thereafter. This finding correlated with the morphologic expression of tubular necrosis, not apoptosis. Hypoxia/reoxygenation caused proximal tubular segment death (44 to 64%), and HK-2 cells were slowly killed by both the H2O2 and the energy depletion/Ca(2+)-loading protocols. However, neither protocol induced ethidium bromide- or tdt-detectable DNA laddering. It was concluded that: (1) minimal DNA laddering develops postischemia, and this change is reliably detected only by the tdt method; (2) it correlates with the morphologic expression of tubular necrosis, not apoptosis; and (3) in vitro oxidative- and energy depletion-mediated proximal tubular cell death can be dissociated from DNA ladder formation.