Alanine protects rabbit proximal tubules against anoxic injury in vitro

Rabbit proximal tubules were incubated aerobically or subjected to anoxia for 30 min followed by 60 min of reoxygenation. The medium contained (in mM) 5 glucose, 10 butyrate, 4 lactate or alpha-ketoglutarate (alpha-KG), and 1 alanine. Anoxic tubules in this medium were severely injured and recovered poorly. If the incubation medium was supplemented with additional alanine (up to 2.5 or 5 mM), then anoxic injury was prevented almost completely. Tubules in high-alanine medium showed modest elevations of ATP during anoxia. Comparable elevations of ATP were induced in anoxic tubules incubated with 4 mM alpha-KG and 5 mM aspartate without alanine. These substrates are metabolized anaerobically in the mitochondria to yield ATP. Surprisingly, anoxic tubules with alpha-KG and aspartate showed severe injury despite elevated ATP. If 5 mM alanine was also present, then additional increments of ATP did not occur, but injury was prevented. Examination of glucose metabolism failed to provide evidence for stimulation of anaerobic fermentations by alanine. These results suggest that alanine-induced cytoprotection during anoxia occurs by mechanisms not related to ATP synthesis, and that elevated ATP in alanine-supplemented tubules may be a result and not the cause of protection. Cytoprotection by alanine was shown to last for less than or equal to 90 min of anoxia. Glycine, a structurally related amino acid, also protects anoxic proximal tubules (J. Clin. Invest. 80: 1446, 1987). The mechanisms that underlie the cytoprotective effects of alanine and glycine remain to be determined.