【背景与目的】

生物钟系统在维持机体外周器官正常的生理功能昼夜节律表达具有重要的意义。本团队前期研究证实肾脏疾病时肾脏生物钟系统被破坏，影响肾脏正常的生理功能，参与血压和尿钠昼夜节律紊乱的分子发病机制。在临床上，肾病患者不仅仅表现为血尿、蛋白尿或肾功能的异常，还出现广泛的肾外脏器功能紊乱，如血脂异常、贫血、骨病及心脑血管并发症，部分还表现为心肾综合征、肝肾综合征等。本研究拟观察在肾病状态下肾外其他外周脏器局部生物钟系统的节律表达情况，进而探讨生物钟系统在肾脏与其他脏器之间的crosstalk对话的媒介作用。

【研究方法】

一、动物实验部分：将雄性sd大鼠（8周龄）随机分为两组，肾病组尾静脉注射阿霉素（6.5 mg/kg），对照组给予等量的生理盐水。监测大鼠动态血压，每4 h每组处死3只大鼠留取血标本和心脏、肝脏组织，测定肝功能、血脂、血糖及血浆肾素活性、醛固酮浓度及血管紧张素浓度变化。应用实时定量pcr方法检测心脏和肝脏核心生物钟基因及下游各脏器特有功能相关钟控基因的mrna表达。应用傅里叶分析合并逐步回归的分析方法进行节律分析。

二、临床上纳入我院肾内科住院肾穿病理提示膜性肾病的中青年患者及健康对照者，监测24h动态血压，同步留取每4h段尿，采用超速离心法提取尿外泌体，应用实时定量pcr的方法检测尿外泌体中核心生物钟基因及肾脏钟控基因enac、ncc和nhe3的mrna表达结果，进行节律分析。

三、从geo数据库中下载数据肾病患者和健康对照外周血单个核细胞和肾组织的基因芯片表达谱数据，利用r软件包对基因表达差异表达分析，并选取生物钟基因及钟控基因进行生物信息学分析。

【研究结果】

动物实验部分：

1、肾病大鼠血压昼夜节律紊乱伴随心脏生物钟系统破坏

（1）对照组正常大鼠的血压具有昼夜节律（p＜0.05），但肾病大鼠收缩压的节律周期完全消失。

（2）对照组大鼠心脏生物钟基因clock、bmal1、cry1、cry2、per1、per2的mrna表达具有昼夜节律的特点（均p＜0.05）。而肾病大鼠心脏cry1基因的节律周期从原有的12h变成（4.8+6）h（p＜0.05），其余的核心钟基因节律消失（均p＜0.05）。

（3）对照组大鼠血浆肾素活性、醛固酮浓度分别呈12h和24h节律（均p＜0.05）；而肾病大鼠血浆肾素、醛固酮表达下降，节律完全消失（均p＞0.05）。

2、肾病大鼠肝脏生物钟及钟控基因节律表达与血脂异常相关

（1）对照组大鼠的tc、tg、ast、alt浓度分别具有周期震荡节律性，分别呈12h、24h，24h和24h的周期节律（均p＜0.05）；而肾病组大鼠的上述节律性紊乱，tg的浓度节律从原有的24h变成12h，而tc、ast、alt的浓度节律表达完全消失（均p＞0.05）；

（2）对照组大鼠的肝脏核心钟基因均出现“昼高夜低”的节律表达，除了基因per1节律为4.8h，其余的钟基因节律均为24h；肾病大鼠核心钟基因cry1和per2仅出现峰值的改变，仍然维持24h的昼夜节律，而其余的钟基因出现明显的节律改变；

（3）肾病大鼠per1钟基因下游肝脏特有血脂代谢、胆固醇转运相关钟控基因lxr、srebp-1、abca1和cyp7表达昼夜节律发生紊乱（均p＜0.05）。

临床研究部分：

1、肾病患者尿外泌体肾脏特异性钟控基因的节律表达研究

（1）健康对照组的尿外泌体中核心生物钟基因的表达具有昼夜节律的特点（均 p＜0.05）。肾病患者尿外泌体中bmal1 基因的表达仍有周期震荡的特点，但节律周期从原有的8h 变成了（4.8+6）h；其余的核心钟基因 clock、cry1、cry2、per1、per2 mrna表达节律完全消失（均 p＜0.05）。

（2）健康对照组尿外泌体中肾脏特有水钠转运相关钟控基因enac、ncc和nhe3 的mrna 表达具有昼夜节律的特点（均 p＜0.05）；节律周期分别为（4.8+12）h、（4.8+8）h和24h；而肾病患者的尿外泌体中的enac、ncc和nhe3昼夜节律完全消失（均p＜0.05）。

2、肾病患者外周血单个核细胞（pbmc）和肾组织钟基因的表达差异分析

（1）对基因芯片数据库分析，膜性肾病患者外周血pbmc的核心生物钟基因clock、bmal1、cry1、per1、dec1和dec2的表达均较健康对照者降低（均p＜0.05），cry2和per2也呈现减少趋势，但无统计学意义（p＞0.05）。

（2）数据库中肾病患者肾组织活检标本和癌旁肾组织标本中核心生物钟基因表达分析，mcd、mn、fsgs患者肾组织除了dec1钟基因的表达与对照组无统计学差异（p＞0.05），其余核心钟基因clock、bmal1、cry1、cry2、per1、per2和dec2表达均升高（均p＜0.05）；钟控基因ncc、enac表达降低，而nhe3、肾素及血管加压素受体基因表达升高（均p＜0.05）；但各个肾病组之间比较并无统计学差异。

（3）生物信息学分析：将心、肝、肾组织钟基因进行聚类分析，构建ppi可视化网络，发现这些基因聚为4大类基因集团，核心生物钟系统为相互间关系最密切的基因集，各脏器自身功能基因交互关系密切，不同脏器功能基因集团之间具有联系。

【结论】

1、肾病状态下，大鼠固有的保守的肾脏、心脏以及肝脏外周组织生物钟系统被破坏，与肾病大鼠呈现的血压、尿钠、转氨酶和血脂的昼夜节律紊乱表现具有协同一致性。肾脏疾病导致肾脏生物钟系统受损，同时累及其他外周脏器生物钟系统和生理功能昼夜节律紊乱，生物钟系统可能为疾病状态下外周组织脏器crosstalk的潜在媒介。

2、健康志愿者尿外泌体中核心生物钟基因及肾脏特异的钟控基因都具有昼夜节律表达的特点，证实监测尿外泌体生物钟基因表达可为我们提供研究人类肾脏疾病时间生物学的简便、可行、可靠手段。肾病患者尿外泌体的核心生物钟基因及肾脏功能钟控基因enac、ncc和nhe3的昼夜节律消失，进一步说明了人类肾脏器质性损害可导致肾脏生物钟系统紊乱。

、数据库中肾病患者pbmc和肾组织中的生物钟系统钟基因表达与正常对照相比也出现了显著的统计学差异，也佐证了在肾病状态下，人外周血和肾组织的生物钟系统出现异常。

、生物信息学分析证实人类生物钟基因编码蛋白与心肝肾功能相关钟控基因编码蛋白存在相关性，生物钟系统可能是肾病状态下心肝肾外周组织脏器之间的crosstalk的潜在媒介。

【关键词】肾病综合征；昼夜节律；外周组织；生物钟基因；crosstalk

background objective:

most physiological functions exhibit circadian rhythmicity. these functional rhythms are driven in part by the circadian clock, which plays an important role in maintaining stability of internal conditions. our earlier study provided that renal local circadian clock system may be involved in occurrence development of chronic kidney diseases(ckd). in clinical, chronic kidney diseases not only present with hematuria, proteinuria abnormal renal function, but also show abnormal blood pressure dyslipidemia, some even manifested as cardiorenal syndrome hepatorenal syndrome or other extra-renal organs damage. the purpose of this article is to observe the circadian rhythm of core clock genes clock controlled genes of extra-renal organs, then explore whether the clock system participates in the crosstalk between peripheral organs in the ckd.

methods:

animal experiments:

sprague-dawley (sd) male rats(8 weeks) were romly divided into adriamycin rats (adrs) group control rats group. adrs were injected 6.5 mg/kg adriamycin via vein to establish nephrotic rats model two weeks later while control rats were injected the equal volume of saline. three rats in each group were sacrificed in six time points to get the blood sample, heart liver tissues, respectively. the mrna expressions of core clock gene specific clock controlled genes in heart liver were evaluated by the real-time quantitative pcr. all the data were analyzed by a partial fourier analysis stepwise regression. the changes of liver function tests, blood lipid, blood glucose plasma renin activity, aldosterone concentration angiotensin concentration were measured.

clinical research:

we investigated the features of 24-hour ambulatory bp monitoring records from ckd patients in comparison with health volunteers in our hospital collected 200ml urine of them every 4-hour, followed the fresh urine exosomes isolated using ultracentrifugation procedures. the mrna expressions of core clock genes kidney-specific clock controlled genes enac、ncc、nhe3 in urine exosomes were evaluated by the real-time quantitative pcr. all the data were analyzed by a rhythm analysis.

microarray data analysis

the gene expression profiles of peripheral blood mononuclear cell (pbmc) samples renal tissue samples were downloaded from gene expression omnibus database. the raw data of clock genes were bioinformatics analysis by r software, string, cytoscape software david database.

results:

animal experiments:

1.1 disturbances of circadian rhythm of blood pressure local cardiac clock genes in nephrotic rats 

(1) in sd controls, the sbp, dbp, map hr appeared 24-hour normal circadian pattern the peak times were all at active period(dark time) (p＜0.05). but the rhythm of sbp was completely disappeared in adr rats(p＜0.05).

(2) in sd controls, cosinor analysis identified daily rhythms period of the core clock genes (clock、bmal1、cry1、cry2、per1、per2) mrna expression in the heart were 4.8+12h, 24h, 12h, 12+24h, 4.8+12h 12h (all p＜0.05),respectively. however, the cardiac timing system of adr rats was disturbed. the cry1 mrna remained circadian rhythm, changed the period from 12h to 4.8+6h, the rhythm of other 5 core clock genes mrna were completely disappeared(p＞0.05).

(3) the plasma aldosterone renin concentration presented 12h 24h daily rhythms in sd rats(p＜0.05). while the diurnal changes completely disappeared in adr rats(p＞0.05).

1.2 disturbances of circadian rhythm of blood lipid local hepatocyte clock genes clock controlled-genes in nephrotic rats

(1) in sd controls, cosinor analysis identified daily rhythms period of the levels of serum tc, tg, ast alt were 12 h, 24 h 24 h (all p＜0.05), respectively. in adr rats, the rhythm of above indicators were disturbed. the rhythm of tc, ast alt were completely disappeared(p＞0.05). while the tg remained circadian rhythm, changed the period from 24h to 12 h.

(2) in sd controls, the local hepatocyte core clock gene of the rats showed the 24h rhythm, except that the per1 rhythm was 4.8h.

(3) the circadian rhythm of the local hepatocyte clock genes cry1 per2 of the nephropathy rats remained the 24h circadian rhythm, but the rest of other 4 core clock genes showed obvious rhythm changes(all p＜0.05).

(4) the liver specific clock-controlled genes for lipid metabolism cholesterol transporting of lxr、srebp-1、abca1 cyp7 expression pattern were also disturbed in adr rats, while these genes exhibit normal circadian patterns in sd control rats (all p＜0.05).

clinical research:

2.1 rhythmic expression of core clock genes kidney-specific controlled genes in human urine exosomes.

(1) in health controls, cosinor analysis identified daily rhythms period of the core clock genes mrna in the urine exsomes were 24h, 8h, 24h, 24h, 24h (4.8+12)h (all p＜0.05),respectively. but the timing system was disturbed in the urine exsomes of mn patients. the bmal1 mrna still remained circadian rhythm, changed the period from 8h to (4.8+6)h, the rhythm of other 5 core clock genes were completely disappeared(p＞0.05).

(2) in health controls, the rhythm of renal specific clock controlled genes for tubular sodium water transporting of nhe3, enac ncc mrna in the urine exsomes were (4.8+12)h, (4.8+8)h 24h(all p＜0.05),respectively. however, these were disturbed in the urine exsomes of mn patients. the rhythm of nhe3，enac ncc mrna were completely disappeared(p＞0.05).

2.2 bioinformatics analysis of gene expression profile data to screen clock genes clock-controlled genes involved in pbmc renal tissues of ckd patients.

(1) extracted the expression values of the clock genes from the geo database, variance analysis was conducted for these genes. the expressions of core clock genes clock, bmal1, cry1, per1, dec1 dec2 were down-regulated in the mn patients’ pbmc samples compared with health controls（all p＜0.05）. the expression of cry1 per1 also presented downward trend without significance（p＞0.05）.

(2) the expression of genes clock, bmal1, cry1, cry2, per1, per2 dec2, but not dec1, were significantly up-regulated in renal biopsy samples of the mcd, mn fsgs patients compared with the expressions in the tumor-adjacent kidney tissues（p＜0.05）. the expressions of kidney specific clock-controlled genes enac ncc decreased, but nhe3, ren avpr genes increased in these patients（p＜0.05）. however, there was no difference between the different kidney disease groups.

(3) the ppi network was constructed by string database showed that  target clock genes could be divided into 4 major categories, core clock genes were the closest relationship there were links between functional genes group of peripheral organs.

conclusions:

1. kidney disease not only causes local renal clock system damage, but also interfere with the clock system physiological circadian dysfunction of other peripheral organs. the clock system may be a potential pathway of the crosstalk among the peripheral organs.

2. monitoring the expression of core clock gene in human urine exsomes was a feasible reliable method for studying rhythm problems of kidney disease. the circadian rhythm of the clock genes above in the urine exsomes of ckd patients was disappeared that identified the kidney timing system disorders in human kidney disease.

3. there were also significant differences in the rhythms of clock genes in pbmcs renal tissues of ckd patients compared with controls from geo database. it supports the clock system is abnormal in human peripheral blood renal tissues of kidney disease.

4. bioinformatics analysis confirmed that the human clock genes coding protein in heart, liver kidney are related to their specific clock-controlled functional gene-dependent proteins, the timing system may be participated in the crosstalk between peripheral organs in the ckd.

key words: nephrotic syndrome, circadian rhythm, peripheral organs, clock genes, crosstalk.