THe long-term objective of this project is to elucidate molecular mechanisms by which a reduction in extracellular potassium (K) concentration is transduced into a mitogenic signal by renal epithelial cells. Kidney growth is induced in rats fed a diet deficient in K, and can be reversed by returning K to the chow. We have shown that cell proliferation, organelle biogenesis, and phospholipid biosynthesis for new cellular membranes in this experimental model can be turned on and off by this simple dietary manipulation. The kidneys of rats fed a K-medium for 1 hour exhibit enhanced glycolysis, activation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD), and accelerated growth. The rapid activation of G3PD is mediated by a cytosolic protein was purified from the cytosol of BSC-1 cells exposed to the low-K mitogenic signal, and subjected to enzymatic cleavage. Amino acid microsequencing of five of the peptide fragments revealed that the modifier is a novel protein. Recent studies utilizing a monospecific antibody revealed that an increased amount of immunoreactive protein and modifier function are present in rat renal papilla and inner stripe of red medulla during the onset of K depletion nephropathy. A new protocol to rapidly purify the modifier has been developed, using an immunoaffinity column, that will facilitate studies aimed at defining the structure and function of this protein.
The specific aims of this revised application are to: 1. use new strategies to obtain a cDNA clone from monkey kidney epithelial cells or rat kidney tissue which encodes the G3PD modifier, and use it to predict the amino acid sequence of the protein; 2. use the cDNA clone to study the regulation of gene expression in normal and proliferation kidney tissue, and 3. use the monospecific antibody to elucidate the contribution of the modifier protein to control of cell growth. Achieving these aims could elucidate the role of the G3PD modifier protein and K in physiological and pathological states.
Showing the most recent 10 out of 19 publications
