?Exposure to cadmium is a long standing problem in human toxicology. A principal size of action is the kidney, where cadmium produces a series of defects in tubular reabsorption of solutes, including glucose, amino acids, and phosphate. Numerous studies identify metallothionein (MT) as a key protein in the cellular speciation and toxicity of Cd2+ as well as in the metabolism of zinc and copper. Bioinorganic research on the metal clusters in MT provide a basis for understanding some of its behavior in cells. Nevertheless, neither the molecular site(s) of renal cadmium toxicity nor the detailed functional roles of metallothionein in subacute Cd toxicity and essential metal metabolism are well understood. In this context, this application sets forth an integrated set of hypotheses and specific aims. It is thought that the two domain structure of metallothionein permits the protein to function simultaneously in intracellular Zn2+ distribution and in Cd2+ sequestration. This idea will be tested in mouse renal tubule cells, using the newly defined defect of Cd2+ to cause specific inhibition of Na+-glucose co-transport without generalized cell toxicity. It will also be explored in model studies on metallothionein in order to understand chemically the cellular properties of the protein. Major questions to be addressed include (1) What are the cellular pathways affected by Cd2+ which lead to inhibition of Na+-glucose co-transport? (2) What is the cellular speciation of Cd2+ in relationship to inhibition of Na+-glucose co-transport? (3) Is the role of metallothionein in zinc metabolism perturbed by cadmium-binding? (4) What is the underlying chemistry of metallothionein that governs its reactivity in cellular zinc and cadmium metabolism and speciation? The proposal involves three senior investigators with complementary expertise, SSB in the culture and examination of the transport properties of kidney cortical cells, CFS in the bioinorganic chemistry of metallo-drugs and -proteins, and DHP in the cellular speciation of essential and toxic metals and the properties of metalloproteins. They will employ radiotracers to follow the effects of Cd2+ on Na+-glucose co-transport, transporter mRNA synthesis, transporter biosynthesis and degradation in relation to Cd2+c distribution. A focus is on its interaction with Zn-MT protein and intracellular distribution of zinc as well as the induction system for MT. Related kinetic experiments on MT and its isolated alpha- and beta-domains will be carried out to model the process of cadmium speciation and localization in cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES004026-07
Application #
3251853
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1987-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Wisconsin Milwaukee
Department
Type
Schools of Arts and Sciences
DUNS #
City
Milwaukee
State
WI
Country
United States
Zip Code
53201
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Tabatabai, Niloofar M; North, Paula E; Regner, Kevin R et al. (2014) De novo expression of sodium-glucose cotransporter SGLT2 in Bowman's capsule coincides with replacement of parietal epithelial cell layer with proximal tubule-like epithelium. J Membr Biol 247:675-83
Meeusen, Jeffrey W; Tomasiewicz, Henry; Nowakowski, Andrew et al. (2011) TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), a common fluorescent sensor for cellular zinc, images zinc proteins. Inorg Chem 50:7563-73
Kothinti, Rajendra; Tabatabai, Niloofar M; Petering, David H (2011) Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA. J Inorg Biochem 105:569-76
Nowakowski, Andrew B; Petering, David H (2011) Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution. Inorg Chem 50:10124-33
Blodgett, Amy B; Kothinti, Rajendra K; Kamyshko, Ivan et al. (2011) A fluorescence method for measurement of glucose transport in kidney cells. Diabetes Technol Ther 13:743-51
Kothinti, Rajendra K; Blodgett, Amy B; Petering, David H et al. (2010) Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: possible reaction of cadmium with the zinc finger domain of Sp1. Toxicol Appl Pharmacol 244:254-62
Ejnik, John; Shaw 3rd, C Frank; Petering, David H (2010) Mechanism of cadmium ion substitution in mammalian zinc metallothionein and metallothionein alpha domain: kinetic and structural studies. Inorg Chem 49:6525-34
Namdarghanbari, Mohammad Ali; Meeusen, Jeffrey; Bachowski, Gary et al. (2010) Reaction of the zinc sensor FluoZin-3 with Zn(7)-metallothionein: Inquiry into the existence of a proposed weak binding site. J Inorg Biochem 104:224-31
Kothinti, Rajendra; Blodgett, Amy; Tabatabai, Niloofar M et al. (2010) Zinc finger transcription factor Zn3-Sp1 reactions with Cd2+. Chem Res Toxicol 23:405-12

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