Mesangial cells (MCs) play a critical role in glomerular biology, both in health and disease. The contraction of these vascular-smooth-muscle- like cells is believed to modulate GFR. The proliferation and secretion of matrix substances by MCs plays a central role in the development of many forms of chronic glomerular disease. The regulation of intracellular pH (pHi), important for a wide variety of cellular functions (enzyme activities, ion conductances, etc.), is expected to be especially critical for MCs. That is because both muscle tension and cell proliferation are exquisitely pH sensitive. This proposal is designed to continue an intensive study of the pH (i) physiologies of rat mesangial cells in primary culture (3rd - 8th passage). Our ultimate goal is to understand how pH(i) is regulated in MCs under control conditions, and after short- and long-term exposure to growth factors that act through different signal-transduction pathways. In previous work, we have shown that MCs use three transporters to regulate pH(i): a Na-H exchanger, a Na+-dependent Cl-HCO3 exchanger (both of which cause pH(i) to increase), and a Na+-independent Cl-HCO3 exchanger (which causes pH(i) to decrease). Furthermore, in work on populations of cells, we have shown that (when assayed at a single pH(i) each of these transporters is activated by mitogens, but that the degree of activation is time dependent. Furthermore, we have developed an approach for accurately assessing the effects of mitogens on the pH(i) dependence of the Na-H exchanger. We propose three major aims: First, to characterie the effects of mitogens and cell shrinkage on the pH(i) dependencies of the three transporters. These experiments will be done on populations of MCs loaded with a pH-sensitive fluorescent dye, and studied in a dual- beam spectrofluorometer. Our approach will be to determine the rates of pH(i) recovery from acid or alkali loads, in the absence and presence of specific inhibitors, and use these data to compute the pH(i) dependencies of the fluxes mediated by each of the three transporters. Second, to use a matematical model of pH(i) regulation to determine if the kinetic descriptions of the transporters (derived in the 1st Aim) account for the pH9I0 vs time records (also obtained in the 1st Aim). Third, to determine the long-range effects of mitogens, and the progression to mitosis, on the activities of each of the three transporters. These experiments will be done on multiple individual MCs loaded with a pH- sensitive fluorescent dye, and studied using a microscope-based digital fluorescence imaging system. We will synchronize our cells, post hoc, based on the time of mitosis; this will allow to determine the time course of how transporter activities change at and near mitosis, which has never before been attempted. The proposed work would lead to the most comprehensive description of pH(i) regulation, and its control by mitogens, in any cell. Moreover, combining the experimental work with the modeling will provide new insights into the factors important for pH(i) regulation. Extending our understanding of pH(i) regulation in MCs both under control conditions and after stimulation by mitogens, could improve our insight into the pathogenesis of glomerular disease.
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