BICARBONATE TRANSPORT BY THE MATURING RENAL TUBULE

主要目的是研究细胞和分子机制。 负责调节肾皮质的酸碱转运 集热管(CCD)。在我们实验室进行的研究表明， 至少有两种功能不同类型的嵌合细胞 在成熟兔的CCD中：一个人通过管腔H+ATPase(型)分泌H+ A)和通过尖端的氯/HCO3交换器(B型)分泌HCO3。A类 细胞表现出快速的顶端内吞活性，而B型细胞则结合 花生凝集素(PNA)。这些特性有助于它们的识别 通过功能性荧光染料。暴露在酸性介质中会导致 间质细胞生理学的改变，其特征是 某些B型电池的尖端Cl2/HCO3交换器及净HCO3的转化 由CCDs从分泌到吸收的运输。尽管A型细胞可以 被刺激以增加H+的分泌以响应酸处理， 这种反应背后的细胞机制还不是很清楚；很少 B类细胞如何反应是已知的。我们已经暗示，它们可能是 能够反转功能极性并分泌H+，但另有选择 解释是可能的，以直接解决这个极性问题，我们计划 使用一种pH敏感的荧光染料，在体外低pH条件下灌流CCDs (BCECF)估计单个嵌入细胞中H+/HCO3的通量 孵化前后。潜在的分子机制 两种亚型间充质细胞的生理学变化 通过确定引发所需的酸刺激的持续时间进行检查 这些变化，以及对蛋白质合成的任何要求，都会发出信号 转导或微丝-微管功能。职能转变 将与荧光免疫细胞化学研究相关联 针对特定的顶端和基底侧端的Cl/HCO3交换器的抗体，以及 H+ATPase，以及酸孵育的Western blotts和2-D蛋白凝胶 Ccd。确定插入的细胞何时具有转运能力 H+/HCO3和对酸负荷的响应，来自中肾、新生儿和 成熟的肾脏将在酸性介质中孵化，并进行检查 如上所述。孤立的ccd，以及次汇流(“未成熟”) 收集的导管细胞系，将通过原位杂交检测 增殖和花纹形成基因的消失以及 酸碱相关(分化)基因的出现。最后，既然 发育中的嵌合细胞可能不表达成熟的表型，它们的 新生大鼠肾脏的谱系将用 逆转录病毒介导的基因转移。这些研究的结果应该会有所帮助。 我们要在细胞水平上更好地了解嵌入细胞是如何 肾脏的发育和分化以及对代谢的反应 酸中毒。他们还应该帮助解释为什么新生儿的能力较差 调节酸碱动态平衡要比成人强。

The main objective is to examine the cellular and molecular mechanisms responsible for the regulation of acid-base transport by the renal cortical collecting duct (CCD). Studies performed in our laboratory have shown that there are at least two functionally different types of intercalated cells in the CCD of mature rabbits: one secretes H+ via a luminal H+ATPase (type A) and one secretes HCO3 via an apical Cl/HCO3 exchanger (type B). Type A cells show rapid apical endocytotic activity, whereas type B cells bind peanut agglutinin (PNA). These properties facilitate their identification by functional fluorescent dyes. Exposure to acidic media results in alteration of intercalated cell physiology, characterized by the loss of apical Cl/HCO3 exchangers by some type B cells and conversion of net HCO3 transport by CCDs from secretion to absorption. Although type A cells can be stimulated to increase H+ secretion in response to acid treatment, the cellular mechanisms underlying the response are not well understood; little is known how the type B cells respond. We have suggested that they might be able to reverse functional polarity and secrete H+, but alternative explanations are possible, To address this polarity issue directly we plan to perfuse CCDs at low pH in vitro, using a fluorescent pH sensitive dye (BCECF) to estimate fluxes of H+/HCO3 in individual intercalated cells before and after the incubation. The molecular mechanisms underlying changes in physiology of both subtypes of intercalated cells will be examined by determining the duration of the acid stimulus needed to set off these changes, as well as any requirement for protein synthesis, signal transduction or microfilament-microtubular function. Functional changes will be correlated with fluorescent immunocytochemical studies using antibodies to specific apical and basolateral Cl/HCO3 exchangers, and H+ATPases, as well as Western blots and 2-D protein gels of acid-incubated CCDs. To determine when intercalated cells become competent to transport H+/HCO3 and respond to acid loading, CCDs from mesonephric, neonatal, and maturing kidneys will be incubated in acidic media and examined as described above. Isolated CCDs, as well as a subconfluent ("immature") collecting duct cell line, will be examined by in situ hybridization for the disappearance of proliferative and pattern-formation genes and appearance of acid-base related (differentiated) genes. Finally, since developing intercalated cells might not express mature phenotypes, their lineage will be determined in the neonatal rat kidney using retrovirus-mediated gene transfer. The results of these studies should help us to better understand at the cellular level how the intercalated cell develops and differentiates and how the kidney responds to metabolic acidosis. They also should help to explain why the newborn is less able to regulate acid-base homeostasis than is the adult.