「日變節律」是視叉上核接受光照訊息，隨外界環境正常的亮暗週期，約略以24小時為週期作同步化規律性循環。而視叉上核是哺乳類動物體內的生物時鐘節律器，透過生物時鐘基因，如Period genes (Per1, Per2 and Per3), Cryptochrome (Cry1 and Cry2), brain and muscle Arnt like protein-1 (Bmal1) and circadian locomotor output cycles kaput (Clock)等，調控許多和日變週期相關的活動。生物時鐘基因不只存在中樞神經，也存在如腎臟等周邊器官。與腎臟功能相關的生理現象如尿液的產生及排除具有日夜的節律性，此外腎臟排除水份、鈉、鉀、鈣、氯、鎂及磷等離子亦然。近來文獻指出生物時鐘基因具調控一些腎功能相關基因，舉例而言，在剔除生物時鐘基因(如Per1 and Clock)小鼠的腎功能相關基因(如vasopressin V2 receptor, aquaporin-4 and alpha subunit of epithelial sodium channel)的表現會受到改變。但生物時鐘基因在腎功能減退時，所扮演的角色目前尚未清楚，所以本論文將探討生物時鐘基因在急性腎衰竭中的表現。本實驗採用雄性C57BL/6小鼠，以腹腔內注射葉酸的方式，誘發急性腎衰竭。實驗進行期間，將小鼠飼養在代謝籠中，每日量測尿量、飲水量及體重變化。使用即時定量聚合酶連鎖反應測量小鼠腎臟中Per1, Per2, Per3, Cry1, Cry 2, Bmal1, Clock, alpha subunit of epithelial sodium channel, sodium/hydrogen exchanger 3, serum/glucocorticoid regulated kinase 1, vasopressin V2 receptor, aquaporin-4 and rennin等基因的表現量。本實驗結果顯示，予小鼠注射葉酸後1天即出現尿量及飲水量增加，在第15天後尿量和對照組無顯著差異，而飲水量直到第21天仍是增加的。體重的變化則是在注射葉酸後1天至第8天，有明顯下降的情形。注射葉酸小鼠的腎組織切片中，觀察到腎小管刷狀緣受破壞及上皮細胞變扁平，顯示有急性腎小管壞死的情形。在鈉離子及水份調節相關的基因方面，發現包括alpha subunit of epithelial sodium channel, sodium/hydrogen exchanger 3, serum/glucocorticoid regulated kinase 1, renin, vasopressin V2 receptor and aquaporin-4等基因表現都有改變。而生物時鐘基因，包括Per1, Per2, Per3, Cry1, Cry 2, Bmal1 and Clock 等基因的表現量及節律性都因受到急性腎衰竭的影響而改變。綜合以上結果，顯示在腎功能減退的情況下，調控腎功能相關基因的生物時鐘基因亦會受影響。

The mammalian circadian timing system is a hierarchically organized network of molecular oscillators driven by the suprachiasmatic nucleus (SCN) of hypothalamus. The molecular basis of circadian timing involves interlocking positive and negative feedback loops which culminate in the rhythmic expression and activity of a set of circadian-clock genes. These genes include the Period (Per1, Per2 and Per3), Cryptochrome (Cry1 and Cry2), brain and muscle Arnt like protein-1 (Bmal1) and circadian locomotor output cycles kaput (Clock). Past studies have shown that the circadian-clock genes present not only in the central nervous system but also in numerous peripheral organs, such as the kidney. Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. Various reports emphasize the importance of circadian-clock genes in the regulation of fluid and electrolyte balance. Recent studies demonstrate that loss of circadian-clock genes result in alteration of renal function. The major focus in this study is to determine the expression of circadian-clock genes in the acute renal failure (ARF). Adult male C57BL/6 mice were intraperitoneally administered folic acid (FA) to induce ARF. The levels of 24-h urinary volume and water intake were measured in individual metabolic cages. The mice were sacrificed at 4-h intervals of the daily cycle, starting at Zeitgeber time 2 (ZT2; time of light-on as ZT0 and light-off as ZT12) on day 3, 10 and 22 after FA injection. We found that the urinary volume and water intake volume began increased, and the body weight was decreased 1 day after FA injection, but the urinary volume and body weight were restored as the control at day 15 and 8, respectively. Histological examination showed disrupted brush borders and flattening of epithelia in the kidneys of FA-treated mice, indicating the occurrence of acute tubular necrosis. In this study, the levels of alpha subunit of epithelial sodium channel, sodium/hydrogen exchanger 3, serum/glucocorticoid regulated kinase 1, vasopressin V2 receptor, aquaporin-4 and rennin expression were decrease in FA-treated mice. The expression of Per1, Per2, Per3, Cry1, Cry2, Bmal1 and Clock were significantly altered not only in mRNA levels but also in circadian patterns in kidney of FA-treated mice. The levels of most gene expression after FA treatment did not restore as the control during the progression of ARF. Impairment in renal function may not only influence the several key regulators of water and sodium but also change the expression of circadian-clock genes. In conclusion, these data suggested that the expressions of circadian-clock genes and renal function-related genes were changed in folic acid nephrotoxicity.

Contents
Acknowledgments Ⅰ
Abstract Ⅱ
Contents Ⅴ
1. Introduction
Circadian rhythm 1
Suprachiasmatic nucleus 1
Transcriptional feedback loops 2
Circadian rhythm and renal function 3
Acute renal failure 4
2. Materials and Methods
Experimental animals and protocols 6
RNA isolation 6
Reverse transcriptase reaction 7
Quantitative real-time polymerase chain reaction 7
Histology 8
Statistical analyses 8
3. Results
Folic acid-induced acute renal failure 10
Expressions of gene related to water regulation and electrolyte balance in the kidney 11
Effect of circadian-clock genes expression to FA-induced ARF in kidney 12
4. Discussion 14
5. Conclusion 18
6. References 28


Figures and Illustrations
Table 1 Polymerase chain reaction (PCR) primers sequences 8
Figure 1 Influence of folic acid (FA) on urinary volume, water intake volume and body weight 19
Figure 2 Renal histology after folic acid (FA) treatment in mice 20
Figure 3 Effects of folic acid (FA) treatment on the daily expression of Na+/H+ exchanger 3 (NHE3)
and alpha subunit of the epithelial sodium channel (αENaC) mRNAs in the kidney 21
Figure 4 Effects of folic acid (FA) treatment on the daily expression of rennin and vasopressin V2 receptor (V2R) mRNAs in the kidney 22
Figure 5 Effects of folic acid (FA) treatment on the daily expression of aquaporin-2 (AQP2) and AQP4 mRNAs in the kidney 23
Figure 6 Effects of folic acid (FA) treatment on the daily expression of serum-and glucose-regulated kinase 1 (SGK1) mRNA in the kidney 24
Figure 7 Effects of folic acid (FA) treatment on the daily expression of Period1 (Per1), Per2 and
Per3 mRNAs in the kidney 25
Figure 8 Effects of folic acid (FA) treatment on the daily expression of Crytochrom1 (Cry1) and Cry2 mRNAs in the kidney 26
Figure 9 Effects of folic acid (FA) treatment on the daily expression of Bmal1 and Clock mRNAs in the kidney 27

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