Cadmium exposure is an important problem in human toxicology. A major site of action is the kidney where Cd2+ causes moderate renal failure including an inability to resorb nutrients such as glucose, phosphate, calcium, and amino acids. The mechanisms that produce this aggregate of toxic effects are not understood. In mouse kidney cortical cells concentrations of Cd2+, which do not affect cell viability, ATP levels, or the activity of the Na+,K+-ATPase, inhibit Na+-glucose and -phosphate cotransport. Further studies have led to the hypothesis that Cd2+ acts directly and specifically on transporter biochemistry not indirectly through disruption of energy conservation mechanisms. It has also been found that metallothionein, the principal site of binding of Cd2+ in cells, can protect against this type of toxicity when present before but not after exposure. To define further both the mechanisms of inhibition of Na+-nutrient transport by Cd2+ and the chemistry and cellular roles of metallothionein related to cadmium exposure, the following specific aims will be explored: (1) To inquire how Cd2+ downregulates the Na+- glucose cotransporter and whether the same type of mechanism applies to inhibition of Na+-phosphate cotransport. (2) To examine whether similar mechanisms of transport inhibition occur in culture and in mice chronically exposed to Cd2+. (3) To determine whether Ca2+-transport is inhibited by Cd2+ in distal tubular cells. (4) To establish the mechanism by which chelating agents remove Cd2+ from kidney cells containing Cd,Zn-MT. (5) To investigate whether the presence of Cd,Zn-MT in kidney cells enhances the toxicity of aminoglycosides. (6) To define the pathway of reaction of apoMT with Cd2+ to form Cd7-MT. (7) To study the mechanism of reaction of apo-MT with Cd-protein complexes. (9) To examine the effect of protein concentration on the reactivity of MT. (8) To define 3-dimensional structure-reactivity relationships in MT. Cultured kidney tubular cells or whole animals will be the subject of experiments in 1-5. Molecular biological and transport methodologies will be used to address the aims in 1-4. Various chemical methods including NMR spectroscopy, radiochemistry, and spectrophotometry will be applied to native MT and its alpha- and beta-domains to investigate aims 5-9.
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