engleski

Cadmium inhibits vacuolar H+-ATPase in rat renal proximal tubule in vivo and in vitro

CADMIUM INHIBITS VACUOLAR H+-ATPase IN RAT RENAL PROXIMAL TUBULE IN VIVO AND IN VITRO Carol M. Herak-Kramberger*, Dennis Brown, and Ivan Sabolic*, IMI, Zagreb, Croatia, and MGH, Boston, USA Nephrotoxicity due to chronic cadmium (Cd) intoxication in humans and experimental animals leads to a defective reabsorption of various compounds by the proximal tubule cells (PTC) and their marked excretion in the urine. The cellular mechanisms affected by Cd in PTC are poorly understood. A vacuolar H+-ATPase, whose function is important for the recycling of membrane componets between the plasma membrane and intracellular organelles via endocytic (EV) and exocytic vesicles, may be a target for Cd. We studied effects of Cd on the vacuolar H+-ATPase in rat kidney PTC in vivo and in isolated organelles in vitro . To induce Cd nephrotoxicity, rats were treated with 2 mg Cd/kg B. W., s. c., for 2 weeks. Immunohistochemical studies in frozen sections of the fixed kidney cortex and Western blot studies in isolated cortical brush-border membrane vesicles (BBMV) showed a strong decrease in abundance of the 31 kD and 70 kD vacuolar H+-ATPase subunits in Cd-treated rats compared to controls. Accordingly, the bafilomycin (BAF) sensitive ATPase activity measured by the Pi-liberation assay in isolated BBMV from Cd-treated and control rats was (mean+SEM) 384+21.8 (n=6) and 248+21.9 (n=9, p<0.001), respectively. In studies in vitro , Cd inhibited the BAF-sensitive ATPase in control BBMV in concentration- and time-dependent manner. As tested by the acridine orange fluorescence quench method, Cd also inhibited the BAF-sensitive intravesicular H+ accumulation in isolated renal cortical EV with mechanisms that involved a) inhibition of the BAF-sensitive H+-pump, b) increase of the vesicle membrane conductance for H+, and c) loss of vesicle integrity. Conclusion: Cd inhibits vacuolar H+-ATPase in PTC organelles in vivo and in vitro and dissipates the transmembrane pH gradiens in intracellular vesicles by increasing their H+ conductance and by damaging their integrity. This may lead to a weaker endocytosis of the filtered proteins and a loss of various brush-border transporters due to impaired recycling of plasma membrane components.

vacuolar H+-ATPase; endocytosis; cadmium nephrotoxicity; kidney; rat

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