CYTOSOLIC CA2+ DEREGULATION AND BLEBBING AFTER HGCL2 INJURY TO CULTURED RABBIT PROXIMAL TUBULE CELLS AS DETERMINED BY DIGITAL IMAGING MICROSCOPY

Acute injury to renal proximal tubule cells has previously been shown to result in elevated cytosolic Ca2+ ([Ca2+]i), blebbing, and eventual cell death. In this study, digital imaging fluorescence microscopy was used to evaluate these changes in response to HgCl2 treatment of cultured rabbit proximal tubular cells. Monolayer cells loaded with fura-2 were treated with 10, 50, or 100-mu-M HgCl2 in both 1.37 mM CaCl2-containing and nominally Ca2+-free (< 5-mu-M) Hanks' balanced salt solution. [Ca2+]i was estimated by measuring the ratio of fluorescent image pairs (collected at 340- and 380-nm excitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blue exclusion. After exposure of cells to 10-mu-M HgCl2, [Ca2+]i initially increased about 2-fold by 5 min; after 50 or 100-mu-M HgCl2, [Ca2+]i rapidly rose 2- to 3-fold, peaked at 1-3 min, and then generally decreased slightly. In nominally Ca2+-free (< 5-mu-M) medium, [Ca2+]i stabilized, but in 1.37 mM Ca2+-containing medium, [Ca2+]i continued to slowly rise, often reaching levels of fura-2 saturation. The rate and extent of blebbing and the rate of cell death were increased in the presence of 1.37 mM Ca2+. These results show that sustained elevations of [Ca2+]i precede both cell blebbing and cell death and that when these elevations are limited by removing extra-cellular Ca2+ the amount of blebbing is reduced and cell viability is prolonged.