Circadian gene profiling in the distal nephron and collecting ducts

Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e. distal convoluted tubule (DCT) and connecting tubule (CNT) and, the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, alphaENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function. Keywords: time course We examined the temporal profiles of gene expression in mouse distal nephron segments and collecting ducts. The RNA was extracted from microdissected distal convoluted tubules and connecting tubules (DCT/CNT samples) or, cortical collecting ducts (CCD samples). Animals were sacrificed for microdissection every 4 hours, i.e. at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 (ZT -- Zeitgeber (circadian) time, indicates time of light-on as ZT0 and time of light-off as ZT12). The microarray hybridization was performed in duplicates on two pools of RNA composed of equivalent amounts of RNA prepared from five animals at each ZT time-point.水与主要电解质的肾脏排泄具有显著的近日节律（circadian rhythm）。这种功能节律性被认为至少部分源于远端肾单位与集合管的分泌、重吸收功能的近日节律变化。本研究探讨了远端肾单位各节段内近日节律的分子机制，所涉节段包括远曲小管（distal convoluted tubule, DCT）、连接小管（connecting tubule, CNT）以及皮质集合管（cortical collecting duct, CCD）。 对显微分离的小鼠DCT/CNT或CCD组织开展时序表达分析后发现，大量与肾脏多种稳态功能密切相关的基因，其表达均呈现显著的近日节律性。该分析还显示，DCT/CNT与CCD均拥有内在的近日计时系统，其特征为核心时钟基因（clock、bma11、npas2、per、cry、nr1d1）以及时钟调控的Par bZip转录因子dbp、hlf与tef的表达均存在强烈振荡。 时钟基因敲除小鼠或dbp/hlf/tef三敲除小鼠的肾脏组织中，多种水钠平衡关键调控因子（血管加压素V2受体、水通道蛋白-2、水通道蛋白-4、alphaENaC）的表达均发生显著改变。从功能层面来看，时钟基因的缺失会引发复杂表型，表现为部分性尿崩症、钠排泄节律紊乱以及血压显著降低。 综上，本研究阐明了分子时钟在肾脏功能中的核心作用。 关键词：时间进程 本研究检测了小鼠远端肾单位节段与集合管内基因表达的时序特征。实验所用RNA提取自显微分离的远曲小管与连接小管（DCT/CNT样本）或皮质集合管（CCD样本）。实验动物每4小时处死一次以进行显微分离，取材时间点为ZT0、ZT4、ZT8、ZT12、ZT16与ZT20（ZT即授时因子时间（Zeitgeber (circadian) time），其中ZT0代表光照开启时刻，ZT12代表光照关闭时刻）。针对每个ZT时间点的5只小鼠等量RNA混合样本，我们开展了两次重复的芯片杂交实验。