Nephron segment and calcium as determinants of anoxic cell death in renal cultures

Abstract

Proximal tubules of the S1, S2 and S3 segments, medullary thick ascending limbs of Henle's loop (MAL) and cortical collecting tubules (CCT) were individually microdissected from rabbit kidneys and cultured for seven days in a hormonally defined media. Anoxia was induced by incubation of cultures in normal medium for 45 min at 25°C in an atmosphere of nitrogen (N2), and cell death was measured by nigrosine dye uptake. Immediately after anoxia, cell death was highest in S3 and MAL segments > S2 > S1 = CCT. The combined effects of anoxia and substrate (glucose, vitamins, amino acid) omission determined after incubation of cultures in phosphate buffered saline containing Ca2+ and Mg2+ (PBS) for 45 min in N2 also showed differential killing dependent on segment of origin: MAL > S3 > S2 CCT > S1. The effects of in vitro 'reflow' were tested by returning cells to their normal oxygenated culture media at 37°C. After the 45 min of anoxia and four to six hr of reflow in normal calcium-containing media, all cells from each segment were dead. Reflow in media lacking calcium for two hr immediately after anoxia then followed by return to normal calcium-containing media was associated with the survival of a significant percentage of cells for 48 hr: S1 (35.3 ± 2.0%), S2 (30.0 ± 2.0%), S3 (46.2 ± 3.0%), MAL (38.7 ± 3.0%), CCT (28.2 ± 2.0%). These results indicate that cells from different nephron segments have different intrinsic susceptibilities to anoxia and that the catastrophic effect on cell survival induced by 'reflow', that is, return of oxygen and substrates, can be significantly attenuated by removal of calcium from the environment for the initial two hr after the insult.