REGULATION OF THE RENAL NA+/CA++ EXCHANGER

肾脏 NA /CA 交换器的调节

• 批准号: 2147832
• 负责人: ROBERT F REILLY
• 金额: $ 13.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2147832
• 关键词: 1,25 dihydroxycholecalciferol; RNase protection assay; calcium flux; calcium metabolism; cellular polarity; human tissue; immunoelectron microscopy; immunoprecipitation; intracellular transport; laboratory mouse; laboratory rabbit; membrane transport proteins; molecular cloning; monoclonal antibody; parathyroid hormones; phosphorylation; protein transport; radiotracer; renal tubular transport; sodium

Renal Ca2+ reabsorption plays an important role in total body Ca2+ homeostasis. The distal tubule (distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD)) reabsorbs only about 10% of the filtered load of Ca2+, but is the major regulatory site for Ca2+ reabsorption in the kidney under the control of parathyroid hormone (PTH). Recently, Bindels et. al. suggested that l,25-dihydroxyvitamin D3 also regulated transepithelial Ca2+ flux. In the distal tubule it is generally accepted that Ca2+ enters the cell across the apical membrane passively down a large electrochemical gradient via calcium channels, but must then exit actively across the basolateral membrane. The exit step can occur through at least two different transport pathways, the Na+/Ca2+ exchanger, and the Ca2+-ATPase. Studies by Shimizu et. al. in isolated perfused tubules (CNT-rabbit) and Bindels et. al. in primary cultures (CNT and CCD- rabbit) suggest that the Na+/Ca2+ exchanger plays a major role in the basolateral exit of Ca2+ in this nephron segment. PTH appears to regulate both the apical entry of Ca2+ and its basolateral exit Gesek et al. in an immortalized mouse cell line from medullary thick ascending limb and DCT, showed that PTH initially stimulated the opening of a chloride channel that hyperpolarized the cell membrane. After a short delay, this was then followed by the opening of a dihydropyridine- sensitive Ca2+ channel that was activated by hyperpolarization. That will directly affects Na+/Ca2+ exchange activity in kidney was reported in basolateral membrane vesicles by several groups. The effect was the result of a change in the Vmax for Na+ gradient-dependent Ca2+ uptake with no change in Km, and was not mimicked by dibutyryl cAMP. Recently, we reported the molecular cloning of a renal Na+/Ca2+ exchanger from rabbit kidney cortex and its immunolocalization using both monoclonal and polyclonal antibodies directed against an intracellular epitope. We showed that the Na+/Ca2+ exchanger is highly homologous to the canine cardiac sarcolemmal Na+/Ca2+ exchanger (95% amino acid identity), and is primarily expressed in the distal nephron (CNT) of both rabbit and rat in this proposal we will use these cDNAs and antibodies to examine the molecular mechanisms responsible for regulation of the renal Na+/Ca2+ exchanger in a primary culture of CNT isolated by indirect immunodissection. These studies will require an antibody directed against an extracellular epitope (for indirect immunodissection). Therefore, a monoclonal antibody will first be generated to an extracellular epitope of the exchanger, and employed to isolate the primary culture. The mechanisms of regulation of the exchanger in response to the acute (1-3 hrs.) and chronic (72 hrs.) exposure to PTH and 1,25-dihydroxyvitamin D3 will then be examined. These studies will provide important novel insights into the molecular mechanisms responsible for the regulation of the renal Na+/Ca2+ exchanger.

肾钙重吸收在体内钙离子总量中起重要作用 动态平衡。远端小管(远端曲管(DCT)，连接 小管(CNT)和皮质集合管(CCD))仅重吸收约10% ，但它是钙离子的主要调节部位 甲状旁腺激素(PTH)控制下的肾脏重吸收。 最近，Bindels et.艾尔建议L，25-二羟基维生素D3也 调节跨血管内皮细胞钙离子流量。在远端小管中，通常 接受Ca~(2+)通过根尖膜被动进入细胞 通过钙通道下降很大的电化学梯度，但随后必须 主动穿过基底膜退出。可以发生退出步骤 通过至少两条不同的转运途径，钠/钙交换器， 和钙-三磷酸腺苷酶。清水等人的研究艾尔在隔离灌流中 小管(CNT-兔)和Bindels等。艾尔在原代培养中(CNT和CD- 表明Na~+/Ca~(2+)交换器在细胞周期中起主要作用。 肾单位段钙离子的基底侧出射。 甲状旁腺素似乎既调节了钙离子的尖端进入，又调节了它的基底侧。 从Gesek等人的出口下来。在一种永生化的小鼠细胞系中 升肢和DCT显示甲状旁腺素最初刺激开放 使细胞膜超极化的氯离子通道。在一小段时间后 延迟，然后是一个二氢吡啶的开张- 被超极化激活的敏感钙通道。那将是 直接影响肾脏Na+/Ca2+交换活性的报道 基侧膜囊泡由几组组成。效果就是结果 NO作用下钠离子梯度依赖性钙摄取的Vmax变化 Km的变化，不能被二丁酰cAMP模仿。 最近，我们报道了一个肾脏钠/钙交换器的分子克隆。 兔肾皮质来源的单抗及其免疫定位 以及针对细胞内表位的多克隆抗体。我们 表明Na~+/Ca~(2+)交换器与犬的同源性很高 心肌细胞膜Na+/Ca~(2+)交换器(氨基酸同源性为95%)，是 兔和大鼠肾远端肾单位(CNT)主要表达 这项提议我们将使用这些DNA和抗体来检测 肾脏Na+/Ca2+调节的分子机制 间接法分离CNT原代培养中的交换子 免疫解剖。这些研究将需要一种直接针对 胞外表位(用于间接免疫解剖)。因此，a 将首先产生针对细胞外表位的单抗 交换器，并用于分离原代培养物。其作用机制 对交易所的调节响应急性(1-3小时)和 慢性(72小时)接触甲状旁腺素和1，25-二羟基维生素D3将 接受检查。这些研究将为我们提供对 肾脏钠/钙调节的分子机制 交易所。