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抗β1肾上腺素受体自身抗体可导致大鼠肾脏损伤

左琳, 赵锐, 王丽, 吕定超, 石山慧, 王可, 李笑, 吕婷婷, 赵荣瑞, 刘慧荣

山西医科大学基础医学院生理学系，太原 030001；山西省儿童医院临床医学检验中心，太原 030001；山西医科大学病理学教研室，太原 030001；首都医科大学基础医学院生理学与病理生理学系，北京 100069

摘要

自从在原发性扩张型心肌病患者血清中发现抗β1肾上腺素受体自身抗体(autoantibodies against the second extracellularloop of β1-adrenoceptor, β1-AABs)以来，自身免疫机制在心血管疾病发病中的作用日渐受到重视。本研究组前期研究显示血清β1-AABs阳性的成年大鼠部分出现尿潜血及尿蛋白阳性，但机制不清。因此，本实验选择直接静脉给入抗体来观察β1-AABs长期存在对大鼠肾脏结构和功能的影响及可能的机制。用人工合成的β1-肾上腺素受体细胞外第二环(β1-AR-ECII)抗原肽段免疫正常成年大鼠以获取β1-AABs。通过观察β1-AABs对乳鼠心肌细胞跳动频率的影响来反映其是否具有生物学活性。目的抗体经纯化后，通过静脉定期注入成年及老年大鼠体内以观察该抗体对大鼠肾脏结构和功能的影响；生化分析仪定期检测大鼠血清中尿素氮(blood urea nitrogen, BUN)、肌酐(creatinine, CR)和尿酸(uric acid, UA)水平，同时尿分析仪检测尿比重、尿潜血、尿蛋白及尿糖的变化情况，以反映肾脏功能的波动；HE和Masson三色染色观察肾脏结构的变化。同时将β1-AR-EGFP质粒转染至HEK293细胞中，观察β1-AABs通过作用于β1-AR对细胞坏死及凋亡的影响。结果显示：成年及老年大鼠血清中BUN、CR、UA的水平随着β1-AABs的不断给入而渐进性升高，BUN/CR比值随免疫的进行呈现下降趋势；成年大鼠在β1-AABs免疫后出现不同程度的蛋白尿、尿潜血及尿糖水平的升高；同时，HE及Masson三色染色显示：β1-AABs免疫24周后，成年及老年大鼠肾脏集合管周围均可见到大量炎细胞浸润，胶原纤维沉积增加。HEK293细胞的研究结果提示，β1-AABs可通过增加细胞caspase-3水平而促进细胞凋亡的发生，而对LDH活性影响不大。由此提示，β1-AABs长期存在可导致成年及老年大鼠肾脏结构和功能的损害。

关键词： 自身抗体; β1-肾上腺素受体; 肾功能不全; 慢性

分类号：R392

Presence of autoantibodies against β1-adrenoceptor aggravates thekidney injury in rats

ZUO Lin, ZHAO Rui, WANG Li, LV Ding-Chao, SHI Shan-Hui, WANG Ke, LI Xiao, LV Ting-Ting, ZHAORong-Rui, LIU Hui-Rong

Deparment of Physiology, Shanxi Medical University, Taiyuan 030001, China； Clinical Inspection Center, Children Hospital ofShanxi, Taiyuan 030001, China； Department of Pathophysiology, Shanxi Medical University, Taiyuan 030001, China； Department of Physiology and Pathophysiology, Capital Medical University, Beijing 100069, China

Abstract

Since the autoantibodies against the second extracellular loop of β1-adrenoceptor (β1-AABs) have been found in the sera ofpatients with idiopathic dilated cardiomyopathy (IDCM), the involvement of autoimmune mechanisms in the pathogenesis of manycardiovascular diseases has extensively been investigated. Our previous study found that urinary occult blood and protein excretionwere frequently found in the rats with positive β1-AABs, but the mechanisms are unclear. Therefore, we infused the β1-AABs into thevein periodically in an attempt to investigate whether β1-AABs could induce morphological and functional changes in the kidneys ofadult and aged rats and explore the possible mechanisms. The synthetic peptide according to the sequences of the second extracellularloop of β1-adrenoceptor (β1-AR-ECII) was used to immunize the adult rats to acquire enough β1-AABs for use. Neonatal rat ventricularmyocytes (NRVMs) culture was used to observe the biological effects of β1-AABs on the beating rate. The purified β1-AABs weretransfused into the vein of rats. The sera level of blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), urinary specific gravity,protein excretion, occult blood and urinary glucose were detected at the different time points by biochemistry and urine analyzers.HE and Masson’s trichrome staining were used to detect the changes in kidney structure of passively immunized rats. Enhanced greenfluorescent protein (EGFP) and β1-AR-EGFP plasmids were transfected into the human embryonic kidney 293 (HEK293) cellsin order to observe the changes in cell injury with the treatment of β1-AABs. It was found that the sera level of BUN, CR and UAincreased gradually and the ratio of BUN to CR decreased progressively with the administration of β1-AABs. The increasing ofproteinuria, urinary occult blood and urinary glucose was detected by urine analyzer in β1-AABs group. By HE and Masson’scoloration, lots of mononuclear cell infiltration and collagen fibers deposition could be observed at the 24th week of immunization.After the treatment of β1-AABs, the caspase-3 activity increased significantly in the HEK293 cells transfected with β1-AR-EGFPplasmids, while no significant changes were observed for lactate dehydrogenase (LDH) activity. The results indicate that long-termpresence of β1-AABs can induce the morphological and functional damage of the kidneys in adult and aged rats.

Key words: Autoantibody; beta 1 adrenergic receptor; renal insufficiency; chronic

收稿日期：2014-01-11　　录用日期：2014-03-11

通讯作者：刘慧荣　　E-mail: liuhr2000@126.com

引用本文：

左琳, 赵锐, 王丽, 吕定超, 石山慧, 王可, 李笑, 吕婷婷, 赵荣瑞, 刘慧荣. 抗β1肾上腺素受体自身抗体可导致大鼠肾脏损伤[J]. 生理学报 2014; 66 (2): 175-185.

ZUO Lin, ZHAO Rui, WANG Li, LV Ding-Chao, SHI Shan-Hui, WANG Ke, LI Xiao, LV Ting-Ting, ZHAORong-Rui, LIU Hui-Rong. Presence of autoantibodies against β1-adrenoceptor aggravates thekidney injury in rats. Acta Physiol Sin 2014; 66 (2): 175-185 (in Chinese with English abstract).