MATURATION OF CATION TRANSPORT IN DISTAL NEPHRON

成长中的受试者处于正K平衡的状态，并具有有限的 排泄K的能力这表明未成熟的肾脏有一个有限的 钾的分泌能力和/或增强的重吸收钾的能力 肾脏钾分泌的调节部位是皮质集合区 风管(CCD)。与观察到的高净K分泌率相比， 从成年动物分离的CCDs和体外微灌流，片段来自 新生动物没有表现出明显的钾转运。然而，同样是这些新生儿 节段吸收钠的速度是成人的一半，这表明 在阳离子传输机制上存在着根本的不同 在新生儿和成人之间。细胞内钾的分泌，由 主细胞，是由一个两步过程决定的：活性钾吸收到 细胞受到Na-K-ATPase和被动扩散的有利 通过心尖K通道的电化学梯度。以确定是否 顶端钾分泌通道的缺乏限制了早期的净钾分泌 LIFE，我们将使用膜片钳分析来比较心尖K 新生和成熟主细胞的电导。以考察是否 K分泌通道缺乏是由于低开放所致 现有信道的概率(P-O)和/或低信道数(N)，我们 下一步将确定新生儿ccd是否暴露于已知因素 增加P-O或N可诱导净K的分泌。两者之间的差异 新生儿脐带血Na和K转运的开始提示质地 跨上皮钠吸收途径的变化 出生后分化。为了测试这一点，我们将比较根尖Na 钠离子的电导(膜片钳分析)、膜转运蛋白 重吸收(氦辉光光度法)和Na-K-ATPase活性(哇巴因- 新生主细胞和成熟主细胞对86Rb的敏感摄取)； 后者的发现将与基底侧向的测量结果相关联。 膜表面积(电子显微镜)。因为中国的净空研究 新生儿表现出明显的钾滞留，我们还将测试 新生儿钾吸收增强可减少净尿的假说 K排泄量。评价青光眼及外眼对钾的吸收能力 髓集合管(OMCD)，我们将测定K的贡献 净吸收率，以管腔到浴缸的单向86Rb通量测量 从成熟动物分离出来的钾在节段中的运输。我们是否应该记录 在生命早期有显著的钾吸收，我们将测试钾的吸收 通过转运蛋白H-K-ATPase与H分泌偶联 免疫细胞化学鉴定为嵌插细胞。那群人 具有H-K-ATPase功能的嵌入细胞将被 确定并测量H-K交换的极性和活性 确定该泵的活动性是否有成熟的变化。这个 这项申请中提出的研究应该有助于我们理解 新生儿脐带血K分泌量受限的生理基础 并对K分泌过程的调节提供了广泛的见解 及其与钠吸收机制的相互关系。

Growing subjects are in a state of positive K balance and have a limited ability to excrete K. This suggests that the immature kidney has a limited capacity for K secretion and/or enhanced ability to reabsorb K. The major regulatory site of K secretion in the kidney is the cortical collecting duct (CCD). In contrast to the high rates of net K secretion observed in CCDs isolated from adult animals and microperfused in vitro, segments from neonatal animals show no significant K transport. Yet, these same neonatal segments absorb Na at a rate half that measured in the adult, suggesting there exists a fundamental difference in cation transport mechanisms between the neonate and adult. K secretion in the CCD, mediated by principal cells, is determined by a two step process: active K uptake into the cell by Na-K-ATPase and passive diffusion down a favorable electrochemical gradient through apical K channels. To determine whether a paucity of apical K secretory channels limits net K secretion early in life, we will use patch clamp analysis to compare the apical K conductances of neonatal and mature principal cells. To examine whether the absence of conducting K secretory channels is due to a low open probability (P-O) of existing channels and/or a low channel number (N), we will next determine whether exposure of the neonatal CCD to factors known to increase P-O or N induces net K secretion. The discrepancy between onset of Na and K transport in the neonatal CCD suggests that qualitative changes in the transepithelial Na absorptive pathway occur during postnatal differentiation. To test this, we will compare the apical Na conductances (patch clamp analysis), membrane transporters active in Na reabsorption (helium glow photometry), and Na-K-ATPase activity (ouabain- sensitive basolateral 86Rb uptake) in neonatal and mature principal cells; the latter findings will be correlated with measurements of basolateral membrane surface area (electron microscopy). Because clearance studies in the neonate indicate significant K retention, we will also test the hypothesis that enhanced K absorption in the neonate reduces net urinary K excretion. To assess the K absorptive capacity of the CCD and outer medullary collecting duct (OMCD), we will determine the contribution of K absorption, measured as unidirectional lumen-to-bath 86Rb fluxes, to net K transport in segments isolated from maturing animals. Should we document significant K absorption early in life, we will test whether K absorption is coupled to H secretion by H-K-ATPase, a transporter immunocytochemically identified in intercalated cells. That population of intercalated cells possessing functional H-K-ATPase will then be identified and the polarity and activity of H-K exchange measured to determine if there is a maturational change in activity of this pump. The studies proposed in this application should help us to understand the physiologic basis for the limited K secretory capacity of the neonatal CCD and provide broad insight into the regulation of the K secretory process and its interrelationship with the mechanism of Na absorption.