Hypoxic Rise in Cytosolic Calcium and Renal Proximal Tubule Injury Mediated by a Nickel-Sensitive Pathway

Abstract

In the kidney, cell injury resulting from ischemia and hypoxia is thought to be due, in part, to increased cytosolic Ca2+ levels, [Ca2+]i, leading to activation of lytic enzymes, cell dysfunction, and necrosis. We report evidence of a progressive and exponential increase in [Ca2+]i (from 245 ± 10 to 975 ± 100 nM at 45 mins), cell permeabilization and propidium iodide (PI) staining of the nucleus, and partial loss of cell transport functions such as Na+-gradient–dependent uptakes of 14C-alpha-methyiglucopyranoside and inorganic phosphate (32Pi) in proximal convoluted tubules of adult rabbits subjected to hypoxia. The rise in [Ca2+]i depended on the presence of extracellular [Ca2+] and could be blocked by 50 μM Ni2+ but not by verapamil (100 μM). Presence of 50 μM Ni2+ also reduced the hypoxia-induced morphological and functional injuries. We also used HEK 293 cells, a kidney cell line, incubated in media without glucose and exposed for 3.5 hrs to 1% O2–5% CO2 and then returned to glucose-containing media for another 3.5 hrs in an air–5% CO2 atmosphere and finally exposed for 1 min to media containing 1 μM Pl. NiCl2 (50 μM) or pentobarbital (300 μM) more than phenobarbital (1.5 mM), when present in the incubation medium during both the hypoxic and the reoxygenation periods, induced significant (P < 0.001) reductions in the number of cell nuclei stained with Pl, similar to their relative potency as inhibitors of T channels. Our findings indicate that hypoxia-induced alterations in calcium level and subsequent cell injury in the proximal convoluted tubule and in HEK cells involve a nickel-sensitive and dihydropyridine insensitive pathway or channel.