Reduced susceptibility of renal slices to oxidative damage.

Precision-cut slices of animal tissue are widely used for pharmacotoxicology, biotrdnsformation and biochemical studies. Tissue slices offer major advantages over other in v i m systems, as the complex heterogeneous architecture of the different cell types that make up the kidney remains intact. During preparation, however, the slices are subjected to oxidative stress and mechanical and physical injury, changes which ultimately limit their viability and useful life-span, especially during incubation for long-term investigations. It is common to preincubate slices for a period of time, but no bysternatic attempt has been made to assess how viability is affected in different buffers, nor what conditions provide optimum function that will help extend the period of slice viability by reducing o r preventing damage. In an attempt to improve the versatility of these preparations for long-term mechanistic investigation we have determined optimal preincubation conditions using 3 culture media supplemented with various antioxidants. Precision-cut renal cortical slices were prepared from male Wistar rats ( 2 0 0 2 5 0 ~ ) using a Krumdieck mechanical tissue slicer [ I ] . Rats were sacrificed by cervical dislocation. Each kidney was removed, decapsulated and stored in ice-cold Krebs-Hepes medium, pH 7.4 which had been gassed previously with 95%02:5%C02. The kidney was cored along the cortico-papillary axis using a 8mm diameter, motor driven coring tool. From the cores, slices (200215pm thick) were prepared in the tissue slicer filled with pre-cooled (+4"C) phosphate buffered saline containing 0.1% (wlv) low melting point agarose. Slices were collected and stored in ice-cold Krebs-Hepes buffer prior to investigation. In the first part of the experiment, renal slices were incubated for up to 2 h at 37°C in the presence of either Hepes-buffered serum-free Dulbecco Minimum Eagles medium and Hams Nutrient mixture F-12 (DMEM-F12) without (-PR) o r with (+PR) phenol red and RPMI-1640 to establish the medium that maintained maximum viability. Tissue viability was assessed every 30 mins using the reduction of 3-(4,5-dimethylthiar~l-2-yl)-2,5-diphenyl tetrazolium (MTT) to formazan [ 21, leakage of intracellular lactate dehydrogenase (LDH) [ 31 and the production of thiobarbituric acid reactive substance (TBARS) [4] as markers of cell viability and oxidative damage. Cell viability was also assessed by the fluorescein diacetate and ethidium bromide staining method [ S ] , that allows viable cells (green) to be contrasted against dead cells (red nuclei) microscopically (390-490 nm excitation and 510-515 nm emission). Incubation of slices in RPMI-1640 for up to 1 h increased viability by over 109% as assessed by the MTT reduction but a significant loss ( P < 0.05) of LDH (Table 1). Both DMEM-F12 with o r without phenol red stabilized the slices for up to 2 h as assessed by the MTT reduction assay. However, over 20%leakage of LDH had occurred in the former within 30 mins of incubation, while less than 2% loss of LDH was observed in the latter up to 90 min of incubation. In subsequent experiments DMEM-F12 without phenol red was chosen as the most suitable preincubation medium as it showed the highest preservation of viability in renal cortical slices (Table 1). There were no changes in TBARS up to 24 h of incubation. Slices were further incubated at 37°C in the presence of either ascorbic acid, a-tocopherol, deferoxamine, mercaptoethane sulphonic acid (MESNA) and reduced glutathione (all at 1mM concentration) for a specific time period. The addition of L-ascorbic acid significantly increased the Table I . Effect of culture media on MTT reduction (percentage) and leakage of LDH (fold increase) over values f o r fresh rend slices incubated for 2 h (mean 2 S D of n = 3 for 4 slices). *P < 0.01