慢性肾脏病循环中FGF23对心房纤维化的促进作用

doi:10.11958/20240171

天津医药 ›› 2024, Vol. 52 ›› Issue (9): 917-923.doi: 10.11958/20240171

慢性肾脏病循环中FGF23对心房纤维化的促进作用

高攀(), 谢冰歆, 周赞东, 刘彤^△()

天津医科大学第二医院心脏科，天津市心血管病离子与分子机能重点实验室，天津心脏病学研究所（邮编300211）

收稿日期:2024-02-02 修回日期:2024-04-16 出版日期:2024-09-15 发布日期:2024-09-06
通讯作者: ^△E-mail：liutongdoc@126.com
作者简介:高攀（1998），女，硕士在读，主要从事慢性肾脏病和心房颤动相关机制方面研究。E-mail：gaopan_47@163.com
基金资助:
国家自然科学基金资助项目(82170327)

Promoting effect of circulating FGF23 on atrial fibrosis in chronic kidney disease

GAO Pan(), XIE Bingxin, ZHOU Zandong, LIU Tong^△()

Department of Cardiology, the Second Hospital of Tianjin Medical University, Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular Disease, Tianjin Institute of Cardiology, Tianjin 300211, China

Received:2024-02-02 Revised:2024-04-16 Published:2024-09-15 Online:2024-09-06
Contact: ^△E-mail：liutongdoc@126.com

高攀, 谢冰歆, 周赞东, 刘彤. 慢性肾脏病循环中FGF23对心房纤维化的促进作用[J]. 天津医药, 2024, 52(9): 917-923.

GAO Pan, XIE Bingxin, ZHOU Zandong, LIU Tong. Promoting effect of circulating FGF23 on atrial fibrosis in chronic kidney disease[J]. Tianjin Medical Journal, 2024, 52(9): 917-923.

摘要/Abstract

摘要：

目的探究慢性肾脏疾病（CKD）循环中成纤维细胞生长因子（FGF）23通过与心房组织成纤维细胞生长因子受体（FGFR）4结合促进心房纤维化的可能机制。方法选择健康雄性SD大鼠22只，随机数字表法抽取14只行5/6肾切除手术并且喂养15周建立CKD模型，剩余8只作为假手术（Sham）组。观察2组体质量、血压、肾功能、超声心动图、心外膜电标测以及病理指标。酶联免疫吸附试验测定2组大鼠循环中的FGF23水平，左心房组织转录组测序寻找差异表达基因。大鼠心房成纤维细胞分为对照组、FGFR抑制剂组、转化生长因子-β（TGF-β）组及TGF-β+FGFR抑制剂组，采用Western blot法检测α-平滑肌肌动蛋白（α-SMA）、胶原蛋白Ⅰ（ColⅠ）以及磷酸化蛋白激酶B（p-AKT）蛋白的表达。结果 CKD组大鼠收缩压、肌酐以及血尿素氮水平升高。心脏电生理检查显示CKD可促进心房颤动及房室传导阻滞的发生。心脏超声提示CKD组左心房内径明显增大，病理染色显示CKD组左心房发生明显纤维化，心外膜电标测提示CKD组大鼠左心房电传导速度明显减慢并且传导异质性明显增加。CKD大鼠循环中的FGF23水平明显增加。Western blot检测发现CKD组大鼠FGFR4表达上调。阻断心房成纤维细胞FGF23/FGFR4信号通路后，纤维化相关蛋白α-SMA、ColⅠ及p-AKT/AKT降低。结论 CKD可能通过诱导心房结构重构及电重构促进房颤的发生，循环中增加的FGF23可能通过与心房组织中FGFR4结合启动下游AKT通路，进而促进心房纤维化。

关键词: 肾疾病, 心房颤动, 纤维化, 心房重构, 成纤维细胞生长因子, 受体, 成纤维细胞生长因子

Abstract:

Objective To explore the possible mechanisms by which fibroblast growth factor (FGF) 23 promoted atrial fibrosis in circulation of chronic kidney disease (CKD) by binding to atrial tissue fibroblast growth factor receptor (FGFR) 4. Methods Twenty-two healthy male Sprague-Dawley (SD) rats were selected. Rats were randomly selected to undergo 5/6 nephrectomy and fed for 15 weeks to establish a CKD model (n=14). The remaining 8 rats were used as the sham group. The sham group (n=8) underwent the same surgery without removing renal tissue. Body weight, blood pressure, renal function, cardiac ultrasound, epicardial electrocardiography and pathological indices were monitored in both groups. Enzyme-linked immunosorbent assay (ELISA) method was used to determine the circulating levels of FGF23 in the two groups of rats. Transcriptomic analysis of left atrial tissue was performed to search for differentially expressed genes. Rat atrial fibroblasts were divided into the control group, the FGFR inhibitor group, the transforming growth factor-β (TGF-β) group and the TGF-β+FGFR inhibitor group. The expression levels of α-smooth muscle actin (α-SMA), collagenⅠ (Col Ⅰ) and phosphorylated protein kinase B (p-AKT) protein were detected by Western blot assay. Results Systolic blood pressure, blood urea nitrogen and creatinine were elevated in the CKD group of rats. Cardiac electrophysiological study showed that CKD could promote the occurrence of atrial fibrillation (AF) and atrioventricular block. Cardiac ultrasound suggested that the internal diameter of the left atrium was significantly increased in rats of the CKD group. Pathological findings showed that the left atrium in the CKD group underwent significant fibrosis, and epicardial electrical markers showed that left atrial electrical conduction velocity was significantly slower and conduction heterogeneity was significantly increased in the CKD group. These changes were accompanied by higher circulating FGF23. Western blot results showed that FGFR4 expression was upregulated in the CKD group. After blocking the FGF23/FGFR4 signaling pathway in atrial fibroblasts, the fibrosis-related proteins α-SMA, Col Ⅰ and p-AKT/AKT were decreased. Conclusion CKD promotes the occurrence of AF by inducing both structural and electrical remodeling. Increased circulating FGF23 promotes atrial fibrosis by activating the downstream AKT pathway binding to FGFR4 in atrial tissue.

Key words: kidney diseases, atrial fibrillation, fibrosis, atrial remodeling, fibroblast growth factors, receptors, fibroblast growth factor

中图分类号:

R541.75

图/表 15

表1 2组大鼠的生化指标及基本特征比较（n=5，$\bar{x}±s$）

Tab.1 Comparison of biochemical indicators and baseline characteristics between the two groups of rats

组别	体质量/g		Cr/ （μmol/L）		BUN/ （mmol/L）		收缩压/ mmHg
SHAM组	485.60±38.28		23.57±8.18		168.09±8.66		114.17±4.78
CKD组	502.80±28.88		58.05±13.57		245.09±19.83		126.17±6.11
t	0.802		4.866^＊＊		7.955^＊＊		3.460^＊＊
组别		平均动脉压/ mmHg		舒张压/ mmHg		心脏质量/胫骨长度/（mg/cm）
SHAM组		100.73±2.31		93.94±3.28		31.20±4.04
CKD组		103.42±8.86		93.68±9.86		44.41±4.46
t		0.657		0.057		4.907^＊＊

表2 2组大鼠电生理学参数比较（n=5，$\bar{x}±s$）

Tab.2 Comparison of electrophysiologic parameters between two groups of rats

组别	QT间期/ms	窦房结恢复时间/ms	房颤持续时间/s
Sham组	41.60±8.29	170.00±8.72	1.16±2.59
CKD组	86.80±10.73	206.40±25.63	10.02±6.56
t	7.451^＊＊	3.007^＊	2.807^＊

图1 2组大鼠电生理检查心电图

Fig.1 Electrophysiological study of electrocardiograms in two groups of rats

图2 2组大鼠不同时间段LAD变化情况

Fig.2 Changes in LAD at various time intervals in two groups of rats

表3 2组大鼠不同时间段LAD比较（n=5，mm，$\bar{x}±s$）

Tab.3 Comparison of LAD in different time periods between two groups of rats

组别	0周	4周	8周	15周
SHAM组	3.96±0.12	3.95±0.36	3.96±0.34	4.00±0.40
CKD组	3.98±0.35	4.63±0.16	4.67±0.38	4.81±0.10
t	0.131	3.909^＊＊	3.128^＊	4.381^＊＊

图3 2组大鼠左心房组织病理学变化（HE染色、Masson染色，×400）

Fig.3 Pathological changes in left atrial tissue of rats in both groups (HE staining, Masson staining, ×400)

图4 Western blot检测2组大鼠左心房组织中α-SMA、Col Ⅰ蛋白表达

Fig.4 The expression levels of α-SMA and Col Ⅰ protein in left atrium of rats from two groups detected by Western blot assay

表4 2组大鼠心房结构重构及电重构参数比较（n=5，$\bar{x}±s$）

Tab.4 Comparison of structural remodeling as well as electrical remodeling parameters of atria between two groups of rats

组别	组织胶原容积分数	α-SMA	ColⅠ	左心房传导速度/（mm/ms）	绝对不均一性/（ms/mm）	不均匀性指数
Sham组	2.29±0.67	0.26±0.07	0.44±0.26	0.82±0.18	3.82±1.39	1.20±0.45
CKD组	5.12±1.20	1.14±0.59	1.58±0.58	0.39±0.09	7.16±2.87	3.28±1.22
t	4.634^＊＊	3.322^＊	4.020^＊＊	4.780^＊＊	2.343^＊	3.578^＊＊

图5 2组大鼠左心房心外膜电标测典型图

Fig.5 Typical diagram of left atrial epicardial electrical markers in two groups

图6 差异表达基因生物信息分析结果

Fig.6 Results of bioinformatic analysis of differentially expressed genes

图7 Western blot检测2组大鼠左心房组织中FGFR4蛋白表达

Fig.7 The expression of FGFR4 protein in left atrium of rats from two groups detected by Western blot assay

表5 2组大鼠循环FGF23水平及心房FGFR4蛋白表达比较（n=5，$\bar{x}±s$）

Tab.5 Comparison of circulating FGF23 levels and atrial FGFR4 protein expression between two groups of rats

组别	FGF23/（ng/L）	FGFR4
Sham组	2 771.53±1 393.28	0.30±0.27
CKD组	8 452.75±1 377.41	0.97±0.37
t	6.484^＊＊	3.239^＊

图8 Western blot检测各组细胞中ColⅠ、α-SMA、p-AKT、FGFR4蛋白表达 A：对照组；B：FGFR抑制剂组；C：TGF-β组；D：TGF-β+FGFR抑制剂组。

Fig.8 Western blot analysis of Col I, α-SMA, p-AKT, AKT and FGFR4 protein expression in each cell group

表6 4组细胞中α-SMA、ColⅠ、FGFR4、p-AKT的蛋白表达水平比较（n=4，$\bar{x}±s$）

Tab.6 Comparison of protein levels of α-SMA, Col I, FGFR4 and p-AKT between four groups of cells

组别	ColⅠ	α-SMA	FGFR4	p-AKT/AKT
对照组	0.46±0.10	0.36±0.18	0.41±0.08	0.79±0.27
FGFR抑制剂组	0.44±0.13	0.41±0.16	0.27±0.20	0.60±0.28
TGF-β组	0.97±0.11^ab	1.22±0.08^ab	1.19±0.39^ab	2.90±0.94^ab
TGF-β+FGFR抑制剂组	0.58±0.25^c	0.72±0.08^c	0.43±0.16^c	0.34±0.20^c
F	22.585^＊＊	37.142^＊＊	12.359^＊＊	20.705^＊＊

图9 FGF23/FGFR4下游通路图 5/6肾切除所致的肾功能异常会使循环中的FGF23增多，FGF23可与成纤维细胞中的FGFR4受体结合启动下游AKT通路促进纤维化，进而促进房颤的发生。

Fig.9 FGF23/FGFR4 downstream pathway illustration

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慢性肾脏病循环中FGF23对心房纤维化的促进作用

Promoting effect of circulating FGF23 on atrial fibrosis in chronic kidney disease

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