Medical College of Wisconsin, Medicine, Milwaukee, WI 53226
SLC5 is a family of membrane proteins that are found in various tissues and are involved in the transport of different nutrients, ions and water. In the kidney, SLC5A1 (SGLT1), and SLC5A2 (SGLT2) are involved in the reabsorption of glucose from the glomerular filtrate. Previous studies have shown that SGLT1 and SGLT2 are two highly related sodium dependent glucose transporters, and that SLC5A4 (SGLT3) has an amino acid sequence identity of 70%-80% to SGLT1 and SGLT2, but it may be a glucose-activated sodium transporter. However, the function of SGLT3 in the kidney has not been studied. The human genome contains only one gene encoding for SGLT3, but mouse and rat express two genes coding for SGLT3a and SGLT3b. Studies on the toxic effects of metal cadmium on mouse kidney cells showed that exposure of these cells to 5µM of cadmium decreased the mRNA levels of SGLT1 and SGLT2 by 35% and 37% respectively, but increased the mRNA levels of SGLT3a and SLGT3b by 400% and 500% respectively. Before we can study the function of SGLT3 in the kidney, it is necessary to determine its protein expression. From the studies done on mRNA, we expected that SGLT3 protein should be expressed in the kidney. We also hypothesized that due to its amino acid sequence homology to SGLT1 and SGLT2, SGLT3 should be found as a membrane protein in kidney cells. To test our hypotheses we performed Western blotting on tissues from rat kidney and on membrane proteins from mouse kidney cells using custom made mouse antibodies to SGLT3a and SGLT3b. Whole kidneys from rat were used for total protein and primary mouse kidney cell cultures were used for membrane proteins. The cells were grown in defined media for seven days until 80% confluent; then the mRNA expression was induced by addition of 5µM cadmium to the media for ~24 hrs. Cells grown in media alone were also used. We collected the cells, and the cell lysate was subjected to centrifugation at 100,000 xg; the resulting pellet was resuspended in buffer. We quantified the protein and loaded 5-10µg onto a tris-glycine gel, where proteins were separated by electrophoresis and transferred onto a PVDF membrane. We incubated the blots with antibodies to mouse SGLT3a and SGLT3b and carried out detection using infrared imaging. For normalization we also detected the expression of a house’keeping protein, Actin. SGLT3a and SGLT3b have a predicted size of 72 kDa. Our experiments showed reactive bands of ~60 kDa for SGLT3a and GLT3b in both, rat tissue samples and membrane proteins from mouse kidney cells. These results support our hypotheses of SGLT3 protein expression in mouse and rat kidney. We also showed that cadmium-exposed cells expressed higher levels of SGLT3a protein, ~400% than control groups, and that SGLT3a antibody had a higher reactivity than SGLT3b. More experiments are needed in order to confirm the expression of the SGLT3 proteins in the kidneys. These involve improving the Western blot conditions in order to reduce background and maximize intensity of signals for quantification. Purification of the custom made antibodies could also improve reactivity and specificity. Finally after identifying the presence of SGLT3 protein in the kidneys, future studies will try to identify the segments within the kidney where SGLT3 is found as well as its functional role.
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