A continuation of research collaboration is proposed between Dr. Dennis Brown, Renal Unit, Massachusetts General Hospital in Boston, USA, and Dr. Ivan Sabolic, institute for Medical Research and Occupational Health in Zagreb, Croatia. The proposed work takes advantage of the complementary expertise of these two investigators to examine the role of cytoskeletal proteins, vacuolar (V)-ATPase and endosomal vesicle trafficking in renal epithelial cells in heavy metal nephrotoxicity. A variety of studies have emphasized the importance of the cytoskeletal network and intracellular vesicle trafficking for the normal polarity and function of epithelial cells. Disruption of cytoskeletal structure and inhibition of the V-ATPase is usually associated with a loss of polarized targeting of various cell membrane transporters and impairment of some specific functions of these cells. In both industrial and underdeveloped countries heavy metals increasingly represent an environmental and health-threatening problem. Some heavy metals, notably cadmium (Cd), lead and mercury, enter the body by food or inhalation in small concentrations, accumulate in various organs, particularly the kidney, and cause cellular damage. In the kidney, the damage of epithelial cells along the nephron is manifested by reabsorptive and secretary malfunctions. Other heavy metals, such as cis-Pt and its derivatives, have been used in the therapy of various tumors and cause a similar impairment of renal epithelial cell function. Whereas the environmental and general toxicological impact on mammalian cells of these heavy metals have been extensively described, their mechanism of action is poorly understood. Experiments on various cells in culture and our preliminary data from experimental animals in vivo indicate the possibility that Cd and other heavy metals cause a very early and profound disruption of the cytoskeleton and inhibit V-ATPase in intracellular vesicles. In combination, this may lead to a significant impairment of intracellular membrane vesicle trafficking and loss of plasma membrane transporters. Thus, the specific aims of this collaborative work are to demonstrate the cascade of intracellular action of heavy metals following their entrance into renal epithelial cells, i.e., a) the initial inhibition of acidification in intracellular vesicles which is followed by b) disruption of the cytoskeleton and impairment of endo- and exocytosis, and c) a selective loss of various transporters in the plasma membrane. A selective loss of mRNAs specific for some membrane transporters takes place at later times. These experiments are designed to study the fundamental mechanisms of nephrotoxic actions of Cd and other heavy metals in renal cells. With this collaboration, Dr. Sabolic, who has developed a strong international reputation as a leader in the field of transport processes in isolated vesicles and is now recognized as an expert in renal cell biology, will open a new frontier of molecular toxicology in his country. The proposed FIRCA award will provide his basic needs for otherwise poorly-funded research in Croatia and will allow important interactions between US and Croatian investigators by introducing new ideas, techniques and a transfer of technology to the developing country.
