engleski

Inhibition ov vacuolar H+-ATPase by cadmium in rat renal brush-border and endosomal vesicles

INHIBITION OF VACUOLAR H+-ATPase BY CADMIUM IN RAT RENAL BRUSH-BORDER AND ENDOSOMAL VESICLES C. M. Herak-Kramberger, D. Brown and I. Sabolic, IMI, Ksaverska 2, 10000 Zagreb, Croatia, and MGH, 149 13th St., Charlestown, MA 12129, USA Chronic cadmium (Cd) intoxication in humans and experimental animals causes nephrotoxicity which is manifested by a defective reabsorption of various compounds in the proximal tubule (PT) and their increased loss in the urine. The mechanisms of Cd-induced cell damage in the PT are poorly understood. A vacuolar, bafilomycin (BAF)-sensitive H+-ATPase (H+-pump) is present in PT brush-border membrane (BBM) and various intracellular vesicles. This ATPase plays a pivotal role in the recycling of various transporters and other membrane componets between the plasma membrane and intracellular organelles via endocytic (EV) and exocytic vesicles. The H+-ATPase may be a target for Cd. Thus, we studied effects of Cd upon vacuolar H+-ATPase in rat kidney PT cells in vivo and in isolated organelles in vitro. Cd nephrotoxicity was induced by treating rats with CdCl2 (2 mg Cd/kg B. W., s. c., daily, for 2 weeks). As measured by the Pi-liberation assay in isolated renal cortical BBM vesicles (BBMV), the BAF-sensitive ATPase activity in Cd-treated rats was 40% lower compared to the activity in control animals. Immunohistochemical studies in frozen sections of the fixed kidney cortex as well as Western blot studies in isolated cortical BBMV showed a strongly decreased abundance of the 70 kDa vacuolar H+-ATPase subunit in Cd-treated rats compared to controls. In studies in vitro, the BAF-sensitive ATPase in BBMV and isolated renal cortical EV was inhibited by Cd in a concentration- and time-dependent manner. However, as measured by the quench method of acridine orange fluorescence, the incubation of isolated EV with Cd resulted in a) inhibition of the ATP-dependent intravesicular H+ accumulation (H+pump) b) enhancement of an H+ conductance in the vesicle membrane, and c) loss of vesicle integrity. We concluded that Cd inhibits the vacuolar H+-ATPase (H+-pump) in the PT cell membrane and intracellular organelles in vivo and in vitro. In addition, Cd dissipates transmembrane pH gradients in intracellular vesicles by increasing the H+ conductance and by damaging the integrity of the vesicle membrane. This may impair vesicle-mediated recycling of the cell membrane components and diminish reabsorptive capacity of the PT luminal membrane due to a loss of specific transporters.

vacuolar ATPase; brush-border; endosomes; cadmium; kidney; rat

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