慢性肾脏病大鼠尿钠昼夜节律紊乱的时间生物学机制初探

发布时间：2018-04-03 12:26

本文选题：肾脏　切入点：昼夜节律　出处：《北京协和医学院》2015年博士论文

【摘要】：[研究背景]肾脏是具有节律的器官,参与调控血压及水盐排泄等生理昼夜节律的形成。临床上慢性肾脏病患者表现出多种节律紊乱如血压节律紊乱、睡眠节律紊乱、不宁腿综合征等。虽其发生机制并不明确,但这些节律紊乱部分在肾移植术后得到改善。越来越多的证据表明,外周组织脏器本身的生物钟系统参与生理节律的形成、稳定与调节。我们前期研究已经证实阿霉素大鼠表现肾病综合征的同时呈现出盐敏高血压和血压昼夜节律紊乱,且协同表现为尿钠排泄昼夜节律的紊乱。本研究进一步尝试探究慢性肾脏病大鼠出现的血压和尿钠昼夜节律紊乱的时间生物学机制,探讨肾脏本身固有的生物钟基因和肾脏特有钟控基因如钠离子转运相关基因在慢性肾脏病中的节律变化及其意义。[研究方法]经SD大鼠尾静脉注射阿霉素建立肾病综合征大鼠模型为肾病组,对照组为注射同体积生理盐水的SD大鼠。适应性饲养2周,严格执行开关灯时间,早8：00开照明灯(记为ZT00：00或CT08：00),晚20：00关灯(记为ZT12：00或CT20：00)。ZT00:00-ZT12:00定义为日间(Light Period), ZT12:00-24:00定义为夜间(Dark Period)。分别收集日夜尿,记录日夜尿量,分测日夜尿钠、尿钾、尿氯的浓度,计算日夜间尿量、尿钠、尿钾和尿氯的排泄率。分别于CT10am,2pm,6pm,10pm, 2am,6am处死对照组和肾病组大鼠各3只,分别取大鼠肾组织、血浆标本,应用实时定量PCR方法检测肾组织钟基因CLOCK、BMAL1、Per1、Per2、Cryl、 Cry2及钟控基因NHE3、aENaC、NCC、Ptges、VlaR、V2R mRNA表达；测定6个时间点大鼠血浆肾素活性、醛固酮和血浆血管紧张素II水平。应用部分傅里叶分析合并逐步回归的分析方法对以上数据进行节律性分析。[研究结果]1、对照组正常大鼠尿量、尿钠及尿钾排泄率呈夜高日低的趋势,肾脏组织内钟基因CLOCK、BMAL1、Per1、 Per2、Cry1、Cry2mRNA均表现昼夜节律(p0.05)及钟基因下游与水盐重吸收和排泄相关的钟控基因NHE3、aENaC、NCC、 Ptges、VlaR、V2R mRNA也表现明显昼夜节律(p0.05),各基因峰值均处于大鼠夜间活动期,与大鼠肾脏水盐排泄的夜高日低的节律一致。2、肾病组大鼠尿钠排泄呈日高夜低的节律(p0.05)。肾脏组织内钟基因BMAL1表达呈24小时和12小时节律,钟基因Cry1表达呈4.8小时节律,钟控基因NCC、V2R表达均呈12小时节律,上述基因昼夜节律峰值均移至日间大鼠休息期(p0.05)。而钟基因CLOCK、Per1、Per2、Cry2及钟控基因NHE3、aENaC Ptges、 VlaR表达昼夜节律均消失(p0.05)。3、对照组SD大鼠外周血浆肾素活性水平呈12小时节律(p0.05),血浆醛固酮浓度水平呈24小时节律(p0.05),血浆血管紧张素II浓度水平无节律(p0.05)；肾病组大鼠血浆肾素活性及醛固酮浓度节律消失(p0.05),而血浆血管紧张素II浓度仍无节律(p0.05)。[研究结论]正常大鼠肾脏固有生物钟基因以及肾脏钟控基因表达的昼夜节律稳定而保守,与正常大鼠尿钠排泄节律具有时相协同性。阿霉素肾病大鼠表现为尿钠排泄的昼夜节律倒置,其肾脏局部的钟基因表达节律紊乱,且钟基因下游与水盐重吸收和排泄相关的钟控基因NHE3, aENaC, NCC, Ptges, VlaR, V2R mRNA的表达时相和节律特点均发生改变。这是首次证实在慢性肾病状态下,大鼠肾脏钟基因系统紊乱,与其血压和尿钠昼夜节律紊乱偶联出现,表明肾脏生物钟分子系统在肾脏正常生理功能的昼夜节律中具有重要作用。
[Abstract]:[background] with the rhythm of the kidney is the organ formation, involved in the regulation of blood pressure and the excretion of salt water and other physiological circadian rhythm in patients with chronic kidney disease. The clinical manifestations of a variety of disorders such as blood pressure circadian rhythm disorder, sleep rhythm disorder, restless legs syndrome. Although its pathogenesis is not clear, but the rhythm section improvement in renal transplantation. More and more evidence that the formation of biological clock peripheral organs themselves participate in physiological rhythm, stability and regulation. Our previous studies have demonstrated that rats with adriamycin and nephrotic syndrome showed salt sensitive hypertension and blood pressure circadian rhythm disorder, and collaborative performance for urinary sodium the excretion of circadian rhythm disorders. This study further attempts to explore the biological mechanism of time chronic kidney disease rats blood pressure and urinary sodium circadian rhythm disorder, the body of the kidney The intrinsic circadian clock genes and kidney specific clock controlled genes such as sodium transport related genes rhythm changes in chronic kidney disease and its significance. Methods] SD rat tail vein injection of adriamycin nephrotic syndrome rat model for nephropathy group, the control group was injected with the same volume of saline. The adaptability of SD rats 2 weeks of feeding, the strict implementation of switch time, 8:00 in the morning lights (ZT00:00 or CT08:00), 20:00 at night lights (ZT12:00 or CT20:00).ZT00:00-ZT12:00 (Light Period) is defined as the day, ZT12:00-24:00 is defined as the night (Dark Period) were collected and recorded. Urine, day and night urine volume, urine test is divided into day and night the sodium chloride concentration, urine potassium, urine, urine sodium, urine volume calculation at night, urine and urine excretion rate of potassium chloride. In CT10am, 2pm, 6pm, 10pm, 2am, 6am were control group and nephritic rats 3 rats in each group were taken from rat kidney Fabric, plasma specimens, detection of clock gene CLOCK, renal tissue using real-time quantitative PCR method for BMAL1, Per1, Per2, Cryl, Cry2 and aENaC, clock controlled genes NHE3, NCC, Ptges, VlaR, V2R mRNA expression; Determination of 6 time points in rat plasma renin activity, and plasma aldosterone angiotensin Zhang Su II level. The application of Fourier analysis analysis combined with stepwise regression analysis. The results of the rhythm of]1 on the above data, the control group of normal rats urine volume, urinary sodium and urinary potassium excretion rate was on the night high low trend, kidney tissue clock gene CLOCK, BMAL1, Per1, Per2, Cry1, Cry2mRNA all day and night rhythm (P0.05) and clock genes downstream and water salt reabsorption and excretion of clock controlled genes NHE3, aENaC, NCC, Ptges, VlaR, V2R and mRNA also showed obvious circadian rhythm (P0.05), the gene in rat nocturnal peak period, and the kidney of rats with salt and water excretion, night high low the Same rhythm.2 nephritic rats urinary sodium excretion was high and low night rhythm (P0.05). Renal tissue in clock gene expression of BMAL1 was 24 hours and 12 hours of rhythm, clock gene Cry1 expression was 4.8 hour rhythm, clock controlled genes NCC, V2R expression showed a 12 hour rhythm, the peak circadian gene all rats to day rest period (P0.05). And the clock genes CLOCK, Per1, Per2, Cry2 and aENaC Ptges clock controlled genes NHE3, VlaR, the expression of circadian rhythm disappeared (P0.05.3), the control group of SD rats peripheral levels of plasma renin activity was 12 hour rhythm (P0.05), plasma aldosterone concentration level a 24 hour rhythm (P0.05), plasma angiotensin II concentration level rhythm (P0.05); nephropathy rats plasma renin activity and aldosterone (P0.05), and the rhythm of plasma angiotensin II concentration was no rhythm (P0.05). The research conclusion of normal rat kidney inherent biological Zhong Ji Because the clock controlled gene expression in the kidney and the circadian rhythm of stable and conservative, and the excretion of urine sodium in rats with normal circadian phase. Sometimes cooperativity in adriamycin induced nephropathy rats showed circadian inversion of urinary sodium excretion, renal local clock gene expression rhythm disorder, and clock genes downstream and water salt reabsorption and excretion the clock controlled genes NHE3, aENaC, NCC, Ptges, VlaR, mRNA and V2R expression rhythm are changed. This is confirmed for the first time in the state of chronic kidney disease, rat kidney system clock gene disorders, their blood pressure and urine sodium circadian rhythm disorder coupling, indicate the important role of circadian rhythm in the kidney the normal physiological function of the kidney biological clock in molecular systems.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692

【共引文献】