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 收集管（CCD）。在我们实验室进行的研究表明， 至少有两种功能不同的插入细胞 在成年兔的CCD中：一个通过腔H+ ATP酶分泌H+（类型 A）和一个通过顶端Cl/HCO 3交换器分泌HCO 3（类型B）。A型 细胞显示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。暴露于酸性介质导致 闰细胞生理学的改变，其特征在于 某些B型细胞的顶端Cl/HCO 3交换器和净HCO 3的转化 通过CCD从分泌到吸收的转运。虽然A型细胞可以 被刺激以增加H+分泌以响应酸处理， 这种反应的细胞机制还没有很好地理解;很少 我们知道B型细胞的反应。我们认为他们可能是 能够逆转功能极性和分泌H+，但替代 为了直接解决这个极性问题，我们计划 使用荧光pH敏感染料在体外低pH灌注CCD （BCECF）以估计单个嵌入细胞中H+/HCO 3的通量 孵育前后。的分子机制 闰细胞的两种亚型的生理变化将是 通过确定酸刺激所需的持续时间来检查， 这些变化，以及蛋白质合成的任何要求， 转导或微管功能。 功能变化 将与荧光免疫细胞化学研究相关， 特异性顶侧和基底侧Cl/HCO 3交换剂的抗体，和 H+ ATP酶，以及蛋白质印迹和2-D蛋白质凝胶的酸孵育 CCD。为了确定何时闰细胞变得有能力运输 H+/HCO 3和对酸负荷的反应，来自中肾、新生儿和 将成熟的肾脏在酸性培养基中孵育，并进行如下检查： 上面描述分离的CCD，以及亚融合（“未成熟”） 收集管细胞系，将通过原位杂交检查 增殖和模式形成基因的消失， 酸碱相关（分化）基因的出现。最后，由于 发育中的闰细胞可能不表达成熟的表型， 将在新生大鼠肾脏中使用 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 让我们在细胞水平上更好地理解 发展和分化以及肾脏如何响应代谢 酸中毒他们也应该有助于解释为什么新生儿的能力较低， 调节酸碱平衡比成人。

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.