This proposal provides a means of laying the groundwork for the candidate's career in quantitative biomedical research. His formal Ph.D. training was primarily in inorganic chemistry with an emphasis on metal-oxygen interactions as models for biological systems. He has developed an interest in biochemical and biomedical research from where he could study these systems from the molecular to the cellular level. In the course of this award period, Dr. Gelasco expects to develop skills in cell biology and biochemistry that are needed, through both didactic and laboratory experience. Then, using these new skills in combination with a chemistry and spectroscopy background he will develop independent research projects investigating the role of enzymatic and signaling events, potentially mediated through biological metal centers, in renal failure pathology. He should be very well suited for a """"""""molecular basis of disease""""""""-type approach toward cell biology pathways and in particular signal transduction, because the combination of chemical, spectroscopic and biochemical techniques should provide an effective arsenal to use toward solving complex biomedical problems. Toward these ends, a research plan has been prepared which should provide ample opportunity to develop new skills and to obtain the theoretical background in cell biology and biomedicine required for Dr. Gelasco's long-term goals. The overall goal of the research proposal is to investigate the effects of indoxyl sulfate on cellular redox-activated pathways, especially those involved in the activation of ERK, because ERK is a major contributing signal intermediary in proliferation of glomerular cells and renal hypertrophy. The major hypothesis to be tested is that the activation of ERK by the uremic toxin indoxyl sulfate is the result of alteration of cellular redox, most likely through the overproduction of reactive oxygen species (ROS), and that the ROS generated are specific for different signaling events in this activation cascade. This project will involve a combination of biophysical, biochemical, cell biological and microphysiometric techniques applied to the investigation of the effects of indoxyl sulfate in these systems. Initial work will involve expanding upon recent observations of ERK activation by indoxyl sulfate with a combination of redox microphysiometry and electron paramagnetic resonance (EPR) spectroscopy to measure the extent of ROS involvement in this process. Redox microphysiometric measurements will probe the redox state of living cells using an electrochemical potentiometric method through a novel technique, which is not available in any other setting. An additional factor that makes this proposal particularly unique is the availability of a new Bruker EPR spectrometer in our lab, which we will use in parallel experiments to measure the production of ROS in indoxyl sulfate-treated mesangial and vascular smooth muscle cells. This biophysical technique will be developed to provide a means not only to determine whether indoxyl sulfate acutely alters the levels of ROS in cultured cells, but to determine which free radical ROS are produced and to precisely measure the levels of ROS produced These methods should then be broadly applicable to studying stimulant-induced signaling events in renal cell pathways.
