AGE-DEPENDENCE OF TOLERANCE TO ANOXIA AND CHANGES IN CYTOSOLIC CALCIUM IN RABBIT RENAL PROXIMAL TUBULES

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury . These may account for the difference in sensitivity to anoxia betwee n certain immature and mature renal cells. To address this question, w e studied the effects of anoxia on cytosolic free Ca2+ concentration ( [Ca2+]i), cell integrity, and transport functions in microdissected pr oximal convoluted tubules (PCT) of <3-week-old (newborn) and >12-week- old (adult) rabbits. Tubules were loaded with 10 mu M fura-2 AM by inc ubation for 60 min at 37 degrees C, and then superfused with isosmotic saline solution gassed with either 95%O-2-5%CO2 (control group) or 95 %N-2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometric ally; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubul es (from resting values of 245 +/- 10 to 975 +/- 100 nM, P <0.001), wh ereas in newborn tubules the rise was significantly less (from resting values of 137 +/- 5 to 165 +/- 5 nM, P < 0.001 between anoxic groups) . Transient exposure to 100 mM potassium chloride, which depolarizes t he PCT cells, induced increases in [Ca2+]i from baseline, to 920 +/- 9 0 nM in tubules from adult and to 396 +/- 16 nM in those from newborn rabbits (P <0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newbor n and adult tubules, but to lower levels in newborn tubules. The respo nse to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic i mmatures tubules did not stain with PI. The sodium-dependent uptakes o f a glucose analogue (C-14-alpha-methyl-glucopyranoside) and phosphate ((32)Pi) were preserved in agarose-filled tubules of newborns after a noxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our re sults suggest that: (1) during anoxia, cell Ca2+ rises to critical lev els in PCTs of adults compared with those of <3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+](o) , presumably voltage-sensitive Ca2+ channels or reversal of Na+-Ca2+ e xchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) seco ndary active transport activity and cellular integrity are well preser ved after anoxia in PCT cells of newborn but not of adult rabbits.