Support is requested to continue studies concerning mechanisms of renal hypertrophy and pH regulation. K+ depletion is a model of renal growth and transport adaptation that can be reproduced in cell culture. Primary cultures of proximal tubule cells isolated with specific monoclonal antibodies to microvillus membranes will be utilized to study effects of K+ deficient media on properties of the proximal cell Na/H exchanger and on cell growth. Single-cell measurements of intracellular pH will be recorded on a microfluorimeter by determining fluorescence of the cytoplasmic pH indicator BCECF. DNA synthesis will be assayed by measurement of 3H thymidine incorporation. Conditioned culture media obtained from cells growing in low K+ environments will be tested for effects on Na/H exchange and DNA synthesis to determine whether proximal cells can produce factors capable of stimulating their own pH regulatory processes and growth. The response of low K+ cells to external modifiers of Na/H exchange, such as epidermal growth factor will be tested, and the role of protein kinase C as mediator of EGF or low K effects will be evaluated. To determine a role for humoral effectors on stimulation of Na/H exchange and DNA synthesis, serum from animals adapted to dietary K deficiency and uninephrectomy will be tested for effects on proximal cells. Growth factors produced by glomerular mesangial cells will be tested for possible effects on pH regulation and growth in proximal cells, effects that would suggest coordination of glomerular-tubular function. Levels of fructose 2,6 bisphosphate and some key gluconeogenic and glycolytic regulatory enzymes will be measured in renal tissue from K+ depleted and uninephrectomized rats to determine a role for hexose bisphosphates in the control of carbohydrate metabolism in these adaptive conditions associated with renal hypertrophy. The regulation of 6-phosphofructo2-kinase will be specifically studied. The effects of EGF and low K+ on the hexose monophosphate shunt will be characterized in proximal cells, to correlate changes in activity of this pathway with renal hypertrophy and pH regulatory mechanisms. These studies will provide information relevant to the regulation of ion transport, cell growth and metabolic processes in pathophysiologic states including acid/base and electrolyte imbalance, renal disease and other states associated with renal growth.
| Stanton, R C; Seifter, J L; Boxer, D C et al. (1991) Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity. J Biol Chem 266:12442-8 |
| Stanton, R C; Boxer, D C; Seifter, J L (1990) Expression of Na(+)-H+ exchange and ATP-dependent proton extrusion in growing rat IMCD cells. Am J Physiol 258:C416-20 |
