MATURATION OF CATION TRANSPORT IN DISTAL NEPHRON

生长期受试者处于正钾平衡状态， 排泄能力K.这表明未成熟的肾脏具有有限的 K分泌能力和/或增强的再吸收K的能力。主要 肾脏钾分泌的调节部位是皮质收集区 导管（CCD）。与高速率的净钾分泌相比， 从成年动物中分离并在体外进行微灌注的CCD， 新生动物没有明显的钾转运。然而，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。K分泌在CCD，介导的 主要细胞，是由两个步骤的过程：主动钾吸收到 细胞通过Na-K-ATP酶和被动扩散向下有利 通过顶端钾通道的电化学梯度。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， 生活中，我们将使用膜片钳分析，比较顶端K 新生和成熟主细胞的电导。研究是否 缺乏传导钾分泌通道是由于低开放性 现有信道的概率（P-O）和/或低信道数（N），我们 接下来将确定新生儿CCD是否暴露于已知因素 增加P-O或N诱导净K分泌。之间的差异 在新生儿CCD的Na和K转运的发病表明，定性 跨上皮钠吸收途径的变化发生在 出生后分化为了验证这一点，我们将比较顶端Na 电导（膜片钳分析），Na+中的膜转运蛋白活性 重吸收（氦辉光光度法）和Na-K-ATP酶活性（哇巴因- 敏感的基底外侧86 Rb摄取）在新生和成熟的主细胞; 后者的发现将与基底外侧的测量相关， 膜表面积（电子显微镜）。因为清除研究 新生儿显示显著的钾潴留，我们还将测试 假设新生儿钾吸收增加会减少净尿 K排泄。为了评估CCD和外部的K吸收能力， 髓集合管（OMCD），我们将确定K 吸收率，测量为单向腔-浴86 Rb通量，至净 成熟动物离体肠段钾转运的研究我们应该记录下 在生命早期显著的钾吸收，我们将测试钾吸收是否 通过H-K-ATP酶（一种转运蛋白）与H分泌偶联 在闰细胞中进行免疫细胞化学鉴定。那部分人口的 具有功能性H-K-ATP酶的插入细胞将被 确定和极性和活性的H-K交换测量， 确定该泵的活动是否存在成熟变化。的 本申请中提出的研究应有助于我们了解 新生儿CCD钾分泌能力有限的生理基础 并为钾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.