Most studies of diabetic nephropathy have not focused on the proximal tubule, although adaptations of this nephron segment to high glucose loads may cause tubular injury. In diabetes, hyperglycemia augments the filtered load of glucose to the tubule, and diminishes the tubule/glucose gradient necessary for facilitative glucose efflux. In vitro, Na+-dependent luminal influx (via Na+-GLUT) is decreased, although in vivo, the mass action effect derived from the high glucose load augments glucose influx. We propose to test the hypothesis that the proximal tubule maintains transtubular glucose flux by adaptations of the basolateral transporters (low Km GLUT 1 and high Km GLUT 2) at the level of gene transcription, resulting in depressed levels of GLUT 1 mRNA and protein, and increased levels of GLUT 2 mRNA and protein. This critical adaptation causes glucose to increase in the tubule due to the higher efflux Km of overexpressed GLUT 2. In this manner, the tubule/glucose gradient (the energy source for facilitative glucose efflux) is restored. However, the tubular glucose (and osmolar) load is increased, which may promote glucose flux through the polyol pathway. We plan to test the hypothesis with time course experiments using the streptozotocin diabetic rat model. We will evaluate the effect of glucose and insulin on the adaptation by measuring changes in GLUT 1 & MRNA, and protein plus GLUT activity in tubules from diabetic rats treated with insulin (lower glucose and higher insulin) and phlorizin (lower glucose and low insulin). The molecular mechanism of the adaptations will be assessed. The regulation of GLUT 1 & 2 gene transcription by tubular nuclear factors will be examined in diabetic rats, with the intent of identifying the trans acting factors responsible for the adaptation. To support the hypothesis, the role of glucose influx and Na+ flux will be examined: Na+ -GLUT mRNA and protein plus Na+ -GLUT function will be measured as a function of time. Moreover, since diabetes also increases Na+ influx in vivo, we will measure Na+ /K+ ATPase (alpha1) mRNA, Na+ pump number and activity. We believe that this information may prove valuable for the development of strategies to arrest diabetic nephropathy.
