Chronic exposure to Cd2+ in humans causes kidney failure that is characterized as a Fanconi syndrome, in which an array of Na+-dependent nutrient transport processes is inhibited. Cd2+ causes a sub-lethal, concentration-dependent reduction of Na+-glucose co-transport in mouse kidney cortical cells that is correlated with a suppression of SGLT1 and 2 mRNA transcription. Results from a variety of sources convincingly argues that phosphorylation signaling links Cd2+ with targets in the SGLT system to inhibit Na+- glucose co-transport. We hypothesize that Cd2+ down-regulates sgltl and 2 gene activity at least in part through the activation of protein kinase C. Direct interaction of Cd2+ with protein kinase C in turn, initiates a signaling cascade that culminates in the phosphorylation and down-regulation of Sp1 and/or SGLT1 and 2 proteins. The signaling pathway is hypothesized to involve mitogen activated kinase (MARK) cascades. Further, we hypothesize that Cd2+ directly displaces Zn2+ from Sp1 and, thereby, contributes to the Cd2+- dependent inhibition of SGLT1 mRNA synthesis. Our overall objective is to understand the cellular mechanism of Cd2+-inhibition of SGLT activity beginning at the molecular biological level and concluding with the definition of the molecular site(s) of binding and action of Cd2+. In the process, basic features of kidney sgltl transcriptional regulation will be addressed.
Our Specific Aims are 1. To ascertain whether the Sp1 binding sites in the sgltl promoter are the critical sites that respond to cellular exposure to Cd2+. 3. To identify changes in phosphorylation state of transcription factor Sp1 as one of the immediate molecular events that links cellular Cd2+ ion to its inhibitory effects on sgltl transcription. 4. To determine the roles of PKC and MAP kinases in the signaling pathway between Cd2+ and SGLT1 mRNA synthesis. 5. To define relevant chemical properties of cellular Cd2+-binding proteins implicated in the cellular mechanism of control of sgltl transcription by Cd2+, particularly Sp1, PKC, and metallothionein. 6. To obtain 3-dimensional structural information about Cd2+-binding domains of Sp1 and protein kinase C. The proposed studies combine molecular biological, chemical, and structural experiments in order to achieve an integrated bio- chemical understanding of the mechanism of Cd2+ inhibition of Na+-glucose co-transport in the kidney.
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