CONTROL OF ACID-BASE TRANSPORTERS BY GROWTH FACTORS & HORMONES IN MESANGIAL CELLS

肾小球系膜细胞（MC）在肾小球生物学中起着关键作用， 健康和疾病。 这些血管平滑肌的收缩 类似细胞被认为调节GFR。 增殖和分泌 基质物质的MC发挥了核心作用的发展， 多种慢性肾小球疾病。 调控 细胞内pH（pHi），对多种细胞 功能（酶活性、离子电导等），预计将 对MC来说尤其重要。 这是因为肌肉紧张和 细胞增殖对pH非常敏感。 这项建议是 旨在继续深入研究大鼠的pH（i）生理学 原代培养的系膜细胞（第3 - 8代）。 我们的最终 目的是了解pH（i）在受控MC中是如何调节的 条件下，并在短期和长期暴露于生长因子后 它们通过不同的信号转导途径起作用。 前几 我们的工作表明，MC使用三种转运蛋白来调节pH（i）： Na-H交换器、Na+依赖性Cl-HCO 3交换器（两者均导致 pH（i）增加），以及Na+-非依赖性Cl-HCO 3交换剂（导致 pH（i）降低）。 此外，在研究细胞群时，我们 已经表明（当在单一pH下测定时（i）这些中的每一个 转运蛋白被有丝分裂原激活，但激活的程度 是时间依赖的。 此外，我们还开发了一种方法， 准确评估有丝分裂原对pH（i）依赖性的影响， Na-H交换器 我们提出三个主要目标：第一， 有丝分裂原和细胞收缩对pH（i）依赖性的影响 三个运输机。 这些实验将在人群中进行 的MCs加载了pH敏感的荧光染料，并在一个双- 光束分光荧光计我们的方法是确定 pH（i）在不存在和存在以下物质的情况下从酸或碱负荷中恢复 特异性抑制剂，并使用这些数据来计算pH（i）依赖性 由三种转运蛋白所介导的通量。 第二，使用 pH（i）调节的数学模型，以确定动力学 对转运体的描述（来源于第一个目标）说明了 pH 9 I 0 vs时间记录（也在第1个目标中获得）。 三是 确定有丝分裂原的长期影响，以及 有丝分裂，对每一个活动的三个转运。 这些 实验将在多个单独的载有pH- 敏感的荧光染料，并研究了使用显微镜为基础的数字 荧光成像系统。 我们会同步我们的细胞，事后， 基于有丝分裂的时间;这将允许确定时间 转运蛋白在有丝分裂时和接近有丝分裂时的变化过程， 从未有人尝试过 拟议的工作将导致 最全面的描述pH（i）调节，及其控制， 任何细胞中的有丝分裂原。 此外，将实验工作与 该模型将为重要因素提供新的见解， pH（i）调节。 扩大我们对MC中pH（i）调节的理解 无论是在对照条件下还是在有丝分裂原刺激后， 提高我们对肾小球疾病发病机制的认识。

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.