REGULATION OF RENAL BICARBONATE AND CHLORIDE ABSORPTION

有显著证据表明NHE-3、安娜/H逆向转运蛋白 异构体，编码近端小管顶膜的一大部分 Na/H逆向转运体活动。本研究的总体目标是 研究调控NHE-3的分子机制。在……里面 目的：L，研究将在三个背景下考察NHE-3的慢性调节， 慢性酸中毒，慢性紧张性增加，以及慢性 细胞的cAMP水平。大部分研究将在 培养的细胞，并将检测对NHE-3活性的影响 这些模型是由：1)由于以下原因引起的mRNA丰度的变化： 转录速率或信使核糖核酸稳定性的变化，2)蛋白质的变化 由于合成速率或稳定性的变化而产生的丰度，3)细胞 贩运，4)NHE-3的翻译后修饰；或5)调控 指一种结合蛋白。在适当的情况下，他将在体内进行研究 以确定细胞培养的结果是否适用于大鼠。 在初步研究中，我们已经证明慢性酸中毒 激活c-src，抑制src家族非受体酪氨酸激酶 防止酸诱导的NHE-3的激活。在目标2中，研究将解决 C-src被激活的分子机制以及c-src的作用 SRC在调节肾酸化中的作用。具体来说，研究将 研究pH对c-src的影响是否仅限于近端小管。 或者发生在其他细胞中，以及这种影响是否对c- Src或也涉及其他src家族的非受体酪氨酸激酶。 然后，研究将检查c-src如何被酸修饰 细胞内的磷酸化、结合和定位。研究将会 解决是否过表达结构性激活的c-src 增加Na/H逆向转运蛋白活性。使用c-src基因或 C-yes基因已经被破坏，我们将研究这些基因的作用 活体肾酸化调节中的蛋白激酶。最后，研究 将讨论c-src在酸诱导的即刻早期是否起作用。 Na/H逆向转运蛋白的基因激活和激素激活。 最后一个目标将阐述血管紧张素II的分子机制。 内皮素对NHE-3活性有调节作用。解决的机制将重点放在 贩运和翻译后修饰。研究还将测量 近曲小管周围环境内的内皮素水平。最后，在老鼠身上 ET-3或ETB的基因已经被破坏，将被用于研究 这一途径在肾功能调节中的作用。

Significant evidence exists suggesting that NHE-3, an Na/H antiporter isoform, encodes a significant fraction of proximal tubule apical membrane Na/H antiporter activity. The overall aim of the present studies is to examine the molecular mechanisms responsible for regulation of NHE-3. In Aim l, studies will examine chronic regulation of NHE-3 in three settings, chronic acidosis, chronic increases in tonicity, and chronic increases in cell cAMP levels. The majority of the studies will be performed in cultured cells, and will examine whether effects on NHE-3 activity in these models are mediated by: 1) changes in mRNA abundance due to either changes in transcription rate or mRNA stability, 2) changes in protein abundance due to changes in synthesis rate or stability, 3) cellular trafficking, 4) posttranslational modification of NHE-3; or 5) regulation of a binding protein. Where appropriate, studies will he performed in vivo in rats to determine whether findings in cell culture apply. In preliminary studies, we have demonstrated that chronic acidosis activates c-src, and inhibition of src family nonreceptor tyrosine kinases prevents acid-induced activation of NHE-3. In Aim 2, studies will address the molecular mechanisms by which c-src is activated, and the role of c- src in the regulation of renal acidification. Specifically, studies will examine whether the effect of pH on c-src is specific for proximal tubule cells or occurs in other cells, and whether the effect is specific for c- src or also involves other src family nonreceptor tyrosine kinases. Studies will then examine how c-src is modified by acid with respect to phosphorylation, binding, and localization within cells. Studies will address whether overexpression of a constitutively activated c-src increases Na/H antiporter activity. Using mice in which the c-src gene or the c-yes gene has been disrupted, we will examine the role of these kinases in the regulation of renal acidification in vivo. Lastly, studies will address whether c-src plays a role in acid-induced immediate early gene activation and in hormonal activation of the Na/H antiporter. The last aim will address the molecular mechanisms by which angiotensin II and endothelin regulate NHE-3 activity. Mechanisms addressed will focus on trafficking and posttranslational modification. Studies will also measure ambient endothelin levels in the proximal tubule. Lastly, mice in which the genes for ET-3 or ETB have been disrupted, will be used to study the role of this pathway in the regulation of renal function.