The applicant is a board eligible nephrologist who hopes to pursue a career in academic nephrology. Her major research interest is mesangial cell growth and the principal focus of this proposal is the elucidation of the mechanisms through which this growth is mediated. There are several glomerular diseases, including diabetic nephropathy, which are characterized at an early point by the proliferation of glomerular mesangial cells. Numerous hormones and growth factors stimulate mesangial cells,and the second messenger systems through which many of these growth factors initiate signals in mesangial cells have been described. One of these signalling systems involves increases in cytosolic calcium concentration. Our preliminary data demonstrates the existence of a gradient between nuclear and cytosolic calcium concentrations. In erythroblasts and hepatocytes, we have noted that different hormones cause variable perturbations in this gradient. In this proposal, using the fluorescent probe fura-2 and a digital video imaging system, we plan to study the effects of several hormones on relative nuclear and cytosolic calcium concentrations. We plan to correlate these changes with mesangial cell proliferation as measured by bromodeoxyuridine incorporation (an immunocytochemical method) into cells incubated with these same hormones. Finally, using a series of calcium/EGTA buffers and varying concentrations of calcium ionophore, we hope to develop a set of experimental conditions by which nuclear and cytosolic calcium can be independently manipulated. Under these conditions, we shall again measure cellular proliferation. We believe that these experiments will provide a greater understanding of the mechanisms by which hormones induce a mitogenic response in glomerular mesangial cells. The primary sponsor, Dr. Leonard Jefferson, is an established researcher in the areas of diabetes mellitus, metabolism and the interaction of hormonal and molecular events. Dr. Kathryn LaNoue, the secondary sponsor, has extensively studied the calcium second messenger system. Dr. Joseph Cheung is an expert in the use of digital video imaging systems and Dr. Hugh English has extensively applied the immunocytochemical techniques noted. All of these individuals are experienced investigators and will provide guidance in the execution of experiments and analysis of the data.
