ROLE OF [CA2+](I) IN LETHAL OXIDATIVE INJURY IN RAT CULTURED INNER MEDULLARY COLLECTING DUCT CELLS

Reactive oxygen metabolites have been implicated in the pathogenesis o f toxic, ischaemic and immunologically mediated renal injury. An incre ase in the cytosolic free Ca2+ concentration ([Ca2+](i)) has been prop osed as a mechanism of oxidative stress-induced cell injury. We used a fluorescence spectrometer and a fluorescence probe to measure the [Ca 2+](i) and viability of rat primary cultured inner medullary collectin g duct (IMCD) cells during oxidative stress induced by 5 mM tert-butyl hydroperoxide (TBHP). Initially, this oxidative stress evoked a small increase in [Ca2+](i) which was followed by a slower sustained increa se from the resting level of 170.8 +/- 38.8 nM to 1490.5 +/- 301.7 nM after 60 min, and this preceded the loss of plasma membrane integrity, measured by the propidium iodide fluorescence method. The elimination of extracellular Ca2+ from the culture medium prevented the TBHP-indu ced [Ca2+](i) increase and improved cell viability Restoration of extr acellular Ca2+ resulted in an immediate and large increase in [Ca2+](i ) and extensive cell death. Verapamil, a Ca2+ channel blocker, inhibit ed the [Ca2+](i) increase and afforded significant protection against cellular injury following exposure to TBHP-induced oxidative stress. E xtracellular acidosis also prevented the increase in [Ca2+](i) and cel l death caused by this oxidative stress. These results are consistent with the hypothesis that oxidative stress-induced IMCD cellular injury may be the result of increased [Ca2+](i) caused, in part, by activati on of voltage-dependent Ca2+ channels.